Processing of Blood culture specimens

Blood culture analysis is one of the most important functions of the microbiology laboratory, as the medical staff relies on the information obtained to aid in the diagnosis of bacteraemia or septicaemia and fungaemia. This can be achieved using either automated system or the traditional non-automated or manual system.

Taking of blood culture samples

This is basically taking a blood sample into a pair of blood culture bottles, aerobic and anaerobic and a further sampling usually two hours apart or more in endocarditis. This increases the yield of positive result and may allow better recognition of contamination. This is usually done as soon as symptoms present, preferably before antibiotics are given, for example shortly after pyrexia spike when bacteria are more likely to be found in the blood stream. The procedure is different in children and infants where specific paediatric blood culture bottles are used. It is ideal that this is done by an experienced member of staff as aseptic technique is very important at this stage to avoid contamination. In most conditions other than endocarditis, bacteraemia is intermittent, being related to the fevers and rigors which occur 30-60 minutes after the entry of organisms into the bloodstream (Ford, 2010).

It is recommended that 20-30ml blood be cultured as volume of blood cultured is the most critical factor in the detection of bloodstream infection, but most modern systems only require approximately 10ml of blood to the two blood culture bottles and a few systems restrict the volume to 5ml. There is a direct relationship between blood volume and yield, with approximately a 3% increase in yield per ml of blood cultured. In neonates, 1-2ml is recommended.

Further pairs of blood culture samples are taken over three days in cases of endocarditis or three blood culture samples over one day if therapy is important. This is usually repeated at 48 hours if the result is negative. These samples are incubated on the Bactec FX Blood culture analyser for 5 days or 7 days for endocarditis patients. Samples must be clearly labelled with full patent details including name and date of birth, hospital/NHS number and attached to a patient test request form containing the clinical details. The addition of full clinical details is vital as it gives laboratory and medical staff information on the likely organisms to be isolated. These clinical details may include sub-acute bacterial endocarditis(SBE), infective endocarditis(IE), endocarditis, native valve endocarditis(NVE), prosthetic valve endocarditis(PVE), vegetative, heart murmur/new murmur, mitral valve and aortic valve.

Transportation of the Blood culture samples.

Blood culture samples should be sent to the laboratory as soon as possible after sampling. They can be stored in an appropriate incubator or kept at ambient temperature before sending. Once samples are received in the laboratory, they are processed immediately by either loading them on the automated culture system or processing them manually.

Manual Blood culture system

Blood culture bottles (aerobic and anaerobic) containing culture medium are used in manual blood culture systems and incubated at the appropriate temperature usually 37 degrees centigrade and incubated for 24 hours, 48hours and 5 days (or 10 days if it is an endocarditic sample). The blood samples are subcultured to blood agar, chocolate agar, Fastidious Anaerobic agar and Chromogenic UTI agar after incubation of 24 hours, 48hours and 5 days (or 10 days if it is an endocarditis sample). An example of manual blood culture system is Biomerieux Hemoline. Growth of organisms is identified by an increase in turbidity of the culture medium and/or the haemolysis of red blood cells. This method is labour intensive and requires the frequent checking of the blood culture bottles in the first 48 hours on receipt of the sample for macroscopic evidence of microbial growth. Blind subculture at 24-48 hours and also at the end of the incubation may pose the risk of contaminating the sample or potential infection risk to the laboratory staff whenever the blood culture sample is opened.

Automated Blood culture system

An automated blood culture system should be able to support the rapid growth of a wide range of pathogenic bacteria including fastidious organisms.

The recent improvement in the automated systems involves the detection of CO₂ which is produced due to glucose metabolism during microbial growth. In BD BACTEC FX blood culture system for example, which is used for the rapid detection of bacteria and fungi in clinical specimens, the increase in the production of CO₂ due to microbial glucose metabolism causes a change in fluorescence or reflectance in a chemical sensor located in the bottom of the bottle. The sensor is monitored by the analyser every ten minutes for an increase in fluorescence which is proportional to the amount of CO₂ present. A positive reading indicates the presumptive presence of viable organisms in the vial. Resins can be added to the medium to neutralise a wide range of antibiotics and enhance the recovery of organisms. This instrument can monitor a total of four hundred vials or blood culture bottles which are arranged in four drawers. The racks are continuously incubated at 36 degrees centigrade and agitated for maximum recovery of organisms.

The Biomerieux BacT/Alert is another automated blood culture system which uses slightly different technology in comparison with the BD BACTEC FX. The sensor is monitored by the analyser every ten minutes for an increase CO₂ production. This increases the concentration of hydrogen ions and decreases the pH thus causing the sensor to become lighter green and eventually yellow.

The culture bottles are usually incubated for a standard five days but in some cases such as endocarditis they are incubated for seven days. This seven days incubation usually applies to fastidious organsims, and those that rarely cause human disease other than endocarditis, for example the HACEK group: Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens and Kingella kingae. Bartonella species are becoming increasingly important causes of endocarditis particularly in AIDS patients.

Blood culture bottle media

Most systems employ different media for the isolation of aerobic and anaerobic organisms, and some media are specifically designed for organisms such as fungi and Mycobacterium species.  A variety of blood culture media are commercially available and have been reviewed. They differ in the types and proportions of various supplements and anticoagulants, volumes of broth, headspace atmospheres and the presence of antimicrobial-neutralising agents.  The aerobic bottle may or may not require transient venting to increase the oxygen content in the headspace for strictly aerobic organisms such as Pseudomonas aeruginosa and Candida albicans.

	Aerobic	Anaerobic	Pediatrics
Ingredients	Quantity	Quantity	Quantity
Processed water	40ml	40ml	40ml
Soybean-Casein digest broth	2.75% w/v	2.75% w/v	2.75% w/v
Yeast extract	0.25% w/v	0.2% w/v	0.25% w/v
Animal tissue digest	0.10% w/v	0.05% w/v	0.10% w/v
Dextrose	0.06% w/v	0.2% w/v	0.06% w/v
Hemin	0.0005% w/v	0.0005% w/v	0.0005% w/v
menadione	0.00005% w/v	0.00005% w/v	0.00005% w/v
Sodium citrate	-	0.02% w/v	-
Thiols	-	0.1% w/v	-
Sodium pyruvate	0.10% w/v	0.1% w/v	0.10% w/v
Saponin	-	0.26% w/v	-
Antifoaming agent	-	0.01% w/v	-
Sodium polyanetholsulfonate (SPS)	0.020% w/v	0.035% w/v	0.020% w/v
Sucrose	0.08% w/v	-	0.08% w/v
Pyridoxal HCL (Vitamin B6)	0.001% w/v	-	0.001% w/v
Non-ionic adsorbing resin	10.0% w/v	-	10.0% w/v
Cationic exchange resin	0.6% w/v	-	0.6% w/v

The media are dispensed with added CO₂ for aerobic vials and CO₂ and N₂ for anaerobic vials. The presence of optimum blood volumes (5-10ml) is beneficial in the recovery of some organisms sensitive to Sodium polyanetholsulfonate (SPS) such as Peptostreptococcus anaerobius, because blood can neutralise the toxicity of SPS.

A blood:broth ratio

The blood:broth ratio of about 1:15 is required to remove the antibacterial effects of normal human blood, but this may be reduced to between 1:5 and 1:10 by the addition of 0.05% sodium polyanethol sulphonate (SPS).  SPS has an inhibitory effect on Neisseria species, anaerobic cocci, Streptobacillus moniliformis and Mycoplasma hominis. The inhibitory effects of SPS may be reduced by the addition of scetic to the broth. Some commercial bottles supplement the medium with materials which improve microbial recovery by adsorbing antimicrobial substances and lysing the white blood cells to release the micro-organisms into the blood-broth mixture.

Neutralisation of antimicrobials

Resins can be added to the media to neutralise a wide range of antibiotics and enhance the recovery of organisms. In addition, the addition of beta-lactamase will help to overcome the effect of beta-lactam antibiotics. 

Refereneces

Brooks G F, Butel J S and Morse S A (1998) Jawetz, Melnick and Adelberg’s Medical Microbiology. Appleton and Lange, Stamford, Cunnecticut, USA

Ford M (2010). Fundamentals of Biomedical Science; Medical Microbiology. Oxford University Press, London.

Erysipelas and Erysipeloid

Erysipelas is a bacterial infection of the dermis and upper subcutaneous layer, usually caused by streptococcus pyogens (β-haemolysis Lancefield Group A streptococcus) but also can be caused by β-haemolysis Lancefield Group G streptococcus and Staphylococcus aureus. It usually affects any part of the skin particularly face, legs and feet. The lesion is well raised, demarcated, red, swollen, tender and may develop vesicles and bullae. Symptoms include headache, fever, fatigue and the rapid enlargement of the skin redness

Erysipeloid is a rare cutaneous inflammatory infection in humans caused by Erysipelothrix rhusiopathiae. It usually affects the hands of persons who handle fish and raw meat. The lesion is raised, violaceous in colour with the absence of pus and usually accompanied by itching and burning. The absence of pus usually helps to differentiate it from Staphylococcal and Streptococcal skin infection. E. rhusiopathiae is a Gram positive rod which grows on blood agar, producing small, transparent glistering colonies and may be α-haemolytic. It also grows on Triple sugar iron agar (TSI) producing hydrogen sulphide turning the TSI butt black. They are microaerophlic and facultative anaerobes. 

Automated Urine Microscopy Analysers

The use of automated system in urine analysis reduces the time and cost used in the manual routine analysis. In addition, automated system analyses a high number of samples in a short time (up to 100 samples per hour). There are a few methods available for automated system which includes the Sysmex UF1000i, Menarini sediMAX and Iris iQ200.

The Sysmex UF1000i method uses the urine flow cytometry technology to detect red blood cells, white blood cells, bacteria, epithelial cells and casts in urine. The automated system dilutes 200µL of the urine sample with citrate buffer solution and cationic surfactant with an addition of 40µL of fluorescent dye to complete the mixture. This mixture is then passed through a sheath of flow which is exposed to a laser beam to detect the particles in the stained urine sample with the fluorescent dye staining the nucleic acid in bacteria. The identification and characterisation of particles are based on the fluorescence intensity, forward scatter light and side scatter light intensity which generates electrical signal patterns by photomultiplier conversion which is then measured.

The Menarini sediMAX is an automated microscopy image based sediment analyser used in urine microscopy. The analyser produces a monolayer of urine sediment by centrifugation in a special cuvette. The sediment is analyzed by a bright field microscope and digital camera to capture and categorize 15 particle images based upon size and shape using image processing software. The digital images can be viewed by a trained laboratory staff with zoom capability. The instrument has a high sensitivity (>80%) in the detection of RBC and WBC but has a low sensitivity (approximately 50%) when compared with manual microscopy.

The Iris iQ200 method uses digital imaging and auto particle recognition to classify and quantify urine particles in uncentrifuged urine based upon size and shape. It classifies images such as WBC, RBC, bacteria, casts, yeast and crystals into categories. The digital images generated are collected, approximately 500 frames per sample, then viewed and classified by a trained laboratory staff. This is usually achieved without the use of manual microscopy.

The advantages and disadvantages of the automated urine systems (Sysmex UF1000i, SediMAX and Iris iQ200) are listed below

Analyser	Advantages	Disadvantages
Sysmex UF1000i	Uses uncentrifuged urine samples. Improved bacteria counting and flags for UTI. Urgent urine samples can be processed by operating the analyser manually. Throughput of 100 samples per hour.	Cannot differentiate clinically significant pathologic casts cell from benign casts. Manual microscopy is carried out on flagged results.
sediMAX	This analyser does not require any reagents. Requires minimal laboratory technician training to interpret images that are similar to manual microscopy. Results may be available for viewing from the stored digital images Throughput of 80 samples per hour.	Yeast is mistaken for RBCs. Epithelial cells are mistaken for WBCs. Particle loss may occur due to the ultracentrifugation of urine samples.
Iris iQ200	Uses uncentrifuged urine samples. Accurately quantifies bacteria. Results may be available for viewing from the stored digital images Throughput of up to 101 samples per hour.	Crystals are mistaken for RBCs. There is no sufficient resolution in order to classify all dysmorphic RBC variants which prompt manual microscopy. Requires considerable laboratory technician training for viewing digital images that are different from manual microscopy.

All you need to know about Zika virus and the outbreak

Zika virus is a member of the Flaviviridae virus family and the Flavivirus genus and spread to people through Aedes mosquito bites. These mosquitoes are the same ones that spread dengue and chikungunya virus. However, unlike the mosquitoes that spread malaria (Anopheles mosquito), they are mostly active during the day, so bed nets offer limited protection against the virus. Zika virus has been linked to thousands of babies being born with underdeveloped brain which is a really worrying situation.

The most common symptoms of Zika virus disease include fever, rash, joint pain, and conjunctivitis (red eyes). The illness is usually mild with symptoms lasting from several days to a week. Severe disease requiring hospitalization is uncommon and deaths are rare (CDC). There is no vaccine as of yet for this virus with only advice from doctors is to rest and drink plenty of fluids. Only one in five people infected are thought to develop symptoms. However, the biggest concern about this virus is its effect on unborn babies in the womb and its link to microcephaly.

According to CDC, microcephaly is a birth defect where a baby’s head is smaller than expected when compared to babies of the same sex and age. Microcephaly is condition where a baby’s head is much smaller than expected. During pregnancy, a baby’s head grows because the baby’s brain grows. Microcephaly can occur because a baby’s brain has not developed properly during pregnancy or has stopped growing after birth, which results in a smaller head size. Microcephaly can be an isolated condition, meaning that it can occur with no other major birth defects, or it can occur in combination with other major birth defects. Babies with microcephaly can have a range of other problems, depending on how severe their microcephaly is. Microcephaly has been linked with problems which include seizures, hearing loss, vision problems, and developmental delay such as problems with speech, intellectual disability, problems with movement and balance and feeding problems, such as difficulty swallowing.

It can be caused by infections such as rubella, Toxoplasmosis, Cytomegalovirus, substance abuse during pregnancy or genetic abnormalities. Although the link with Zika virus has not been confirmed, but some babies who died had the virus in their brain and it has been detected in placenta and amniotic fluid too.

It was first identified in monkeys (rhesus macaque) in Zika Forest of Uganda in 1947. The first human case was detected in Nigeria in 1954 and isolated from humans during studies conducted in 1968 and 1971–1975 with outbreaks in some part of Africa, South East Asia and the Pacific Islands since then. Most were small and Zika has not previously been considered a major threat to human health. But in May 2015 it was reported in Brazil and has seen spread rapidly. Brazil had fewer than 150 cases of microcephaly in the whole of 2014, but there have been more than 3,500 reported cases since October. It has since also been reported in Barbados, Bolivia, Colombia, the Dominican Republic, Ecuador, El Salvador, French Guiana, Guatemala, Guadeloupe, Guyana, Haiti, Honduras, Martinique, Mexico, Panama, Paraguay, Puerto Rico, Saint Martin, Suriname and Venezuela. The US National Institute of Health said that it is currently an explosive pandemic re-emergence. WHO expects Zika virus to spread throughout the Americas, however, other scientists have warned that the outbreak could reach Asia countries.

As a preventive measure, the CDC has advised pregnant women not to travel to affected areas. People are also adviced to use insect repellents, cover up with long-sleeved clothes and keep windows and doors closed.

Rubella screening in pregnant women to end in England – BBC

The screening of Rubella in women during pregnancy will end in England in April 2016. This measure is also being considered by rest of the UK. This is according to Public Health England who reported that the success of MMR vaccination meant that the infection was now incredibly rare and the screening was causing unnecessary stress.

Rubella infection during pregnancy can lead to brain damage and other health problems for the baby in the womb. The viral infection which is also known as German measles is mild, causing a rash and fever. But it becomes very serious in the first 20 weeks of pregnancy, when it can lead to sight and hearing problems, brain damage and heart defects.

Screening during pregnancy

During pregnancy, women are offered a blood test to determine whether they are immune to the virus. In 2012, the United Kingdom National Screening Committee recommended that rubella screening should stop because levels of the virus were so low that the World Health Organization classed rubella as eliminated in the UK. Public Health England (PHE) said that high uptake of the MMR jab, which protects against measles, mumps and rubella (MMR) was allowing it to make the decision.

Dr Anne Mackie, the director of screening programmes at PHE, said that the decision to end rubella susceptibility screening in pregnancy in England is based on a rigorous assessment of the evidence and expert clinical advice. Screening for rubella in pregnancy does not give any protection to the unborn baby in that pregnancy. The best preventative measure a woman can take to protect herself from rubella is to ensure she is immunised with the MMR vaccine before she gets pregnant.

The charity Sense, set up by parents affected by rubella, welcomed the news. Joff McGill, from the charity, said: It is a cause for celebration that in the UK there is no endemic rubella and congenital rubella syndrome births are now rare thanks to the childhood immunisation programme and high levels of MMR uptake. Stopping rubella screening in pregnancy will not lead to an increase in cases of congenital rubella syndrome. The only way that will happen is if there are significant rubella outbreaks, and we can ensure that doesn't happen by continued high levels of uptake of MMR.