Tuesday, 29 December 2015

France sees bird flu cases double in less than a week

There has been cases of bird flu in France which has doubled since Friday as reported by the Ministry of Agriculture with a total of 61 on 22 December, 2015 compared with 30 at the end of previous week. In a statement, the Ministry of Agriculture said the 61 cases now covered six regions in south-west France, with the Haute-Pyrenees department the latest to have recorded cases close to the Spanish border.
although the French Minister for Agriculture Stephane Le Foll said that the outbreak was under control, farmers in the Landes region, which has 27 cases, have now lost tens of thousands of birds. However, the sequencing of the H5N1 strain detected in the first outbreak in the Dordogne at the end of November has confirmed the absence of key markers, meaning there is no danger to humans, the ministry confirmed.
According to the latest analysis published by the Animal Plant Health Agency (APHA), the risk of bird flu spreading from France to the UK remains low but heightened. APHA Head of Virology, Professor Ian Brown and Dr Helen Roberts, of APHA’s international disease monitoring team said the risk to the UK as a result of the French outbreaks was primarily around pathways which involve lapses in biosecurity or trade routes, rather than through wild birds. The pair said the French outbreaks were wholly of European origin.
The situation in France simply reinforces what we already know that low pathogenic avian influenza viruses [LPAI] circulate in wild birds and cause occasional spillover outbreaks in poultry. These may be difficult to detect in domestic waterfowl species and they may then mutate into [highly pathogenic avian influenza (HAPI)] viruses. The cases in a France may be related but whether the index case for LPAI and HPAI mutation has been found is unclear.
In the run-up to Christmas, the agency reminded poultry keepers to maintain high standards of biosecurity, remain vigilant and report any signs of animals showing sickness.

Sexually transmitted diseases has exploded in recent years – CDC

Maggie Fox from NBC News has reported that common sexually transmitted diseases such as syphilis and gonorrhoea have exploded in recent years in USA, in part because of reduced funding for public health clinics. There are more than 1.4 million reported cases of Chlamydia last year which is the highest number of cases of any disease ever reported to the Centres for Disease Control and Prevention (CDC).  
CDC reported that the number of syphilis cases reported in 2014 were under 20,000, the highest rate since 1994 and a 15% increase over 2013. They also found 458 cases of syphilis in newborn babies which is a startling 27.5%t increase over 2013. In addition more than 350,000 cases of gonorrhoea were reported which is up 5% from 2013. "Certainly, this is the first time since 2006 that all three of our notifiable sexually transmitted diseases have increased," said the CDC's Dr. Gail Bolan. "Some of the increases are quite alarming." Most of the increases have been seen in young adults, who get infected soon after they first begin having sex.
The CDC estimates that half of the 20 million new sexually transmitted infections that occur every year are among people aged 15-24. Dr Bolan noted that Young people are the most vulnerable and women can lose their reproductive health for a lifetime from infection of Chlamydia or gonorrhoea. While antibiotics can treat the infections, they often do not cause symptoms until damage has been done.
What's going on to cause the increase? There are several factors, the CDC says. Budget cuts are a big factor.  Most recently, there have been significant erosions of state and local STD control programs. Most people don't recognize that the direct clinical care of individuals with sexually transmitted diseases is supported by state and local funds and federal funds. Just one example in October, the Illinois Department of Health stopped paying for STD tests at 100 jails and local health departments across the state. Dr Bolan acknowledged that in one year 7% of local health departments said they closed their STD clinics. And 43% said they had to cut back on the hours they could stay open. About a third had to raise fees and co-pays, something that's been shown to keep some people away.
Another big factor is a change in behaviour among gay and bisexual men. The increase in syphilis among gay men is concerning because we have been seeing this increase for almost a decade, Dr Bolan said. It seems to correlate with the advent of HIV treatment. Dr Bolan also added that HIV treatment is not responsible for the change. HIV infection has become a chronic disease that can be managed with the cocktails of powerful HIV drugs that are now available, instead of a death sentence. HIV patients know they can stay healthy if they take the drugs, and that they are less likely to infect someone else and people are excited about it. And some may have stopped using condoms so consistently, because they are no longer afraid of a deadly infection. Unfortunately, HIV treatment has no impact on prevention of other STDs. The non consistent and correct use of condoms is putting people at risk for STDs.
Uninfected people can also take HIV drugs to protect themselves from infection. That might make people think they're even safer from HIV. Researchers across the country found that people at high risk of HIV who took the drugs in a practice called pre-exposure prophylaxis or PrEP almost never caught HIV, but they did catch syphilis and gonorrhoea. There is some data suggesting that there is less condom use in some populations now. As for Chlamydia, Dr Bolan thinks it's mostly a matter of better reporting that's driving the record numbers of reported cases. Chlamydia has been a very common sexually transmitted infection for years.

- Centers for Disease Control and Prevention (CDC)

Monday, 28 December 2015

Rise in untreatable multi-antimicrobial resistant gonorrhoea strain – BBC

England chief medical officer has warned that Gonorrhoea could become untreatable due to emergence of multi antimicrobial resistant strains termed "super-gonorrhoea" according to BBC
All GPs and pharmacies have been written to by Dame Sally Davies to ensure they are prescribing the correct drugs after the rise of "super-gonorrhoea" in Leeds. This warning came after concerns were raised that some patients were not getting both of the antibiotics needed to clear the infection. Sexual health doctors said gonorrhoea was "rapidly" developing resistance.
in March 2015, a highly drug-resistant strain of gonorrhoea was detected in the north of England. That strain is able to shrug off the antibiotic azithromycin, which is normally used alongside another drug, ceftriaxone.
In Dame Sally Davies letter, the chief medical officer said: "Gonorrhoea is at risk of becoming an untreatable disease due to the continuing emergence of antimicrobial resistance." But while an injection of ceftriaxone and an azithromycin pill are supposed to be used in combination, this may not always be the case for all patients.
The British Association for Sexual Health and HIV (BASHH) warned earlier this year that some online pharmacies were offereing oral medication options only. 
Using just one of the two drugs makes it easier for the bacterium to develop resistance. The letter, which is also signed by chief pharmaceutical officer Dr Keith Ridge, stated: "Gonorrhoea has rapidly acquired resistance to new antibiotics, leaving few alternatives to the current recommendations. "It is therefore extremely important that suboptimal treatment does not occur."
Gonorrhoea is caused by the bacterium called Neisseria gonorrhoeae. The infection is spread by unprotected vaginal, oral and anal sex. Symptoms can include a thick green or yellow discharge from sexual organs, pain when urinating and bleeding between periods. Often the person has no symptoms, however, but can still easily spread the disease to others. Untreated infection can lead to infertility, pelvic inflammatory disease and can be passed on to a child during pregnancy.
Gonorrhoea is the second most common sexually transmitted infection in England and cases are soaring. The number of infections increased by 19% from 29,419 in 2013 to 34,958 the following year.
Dr Jan Clarke, the president of BASHH, told the BBC News website: "We're really pleased that the chief medical officer has stressed that gonorrhoea needs this approach to treatment due to the rapid development of resistance."We need to protect what we've got and we need to encourage pharmacists and general practitioners to follow first-line treatment."
Dr Andrew Lee, from Public Health England, added: "Investigations are ongoing into a number of cases of anti-microbial resistant gonorrhoea. "Public Health England will continue to monitor, and act on, the spread of antimicrobial resistance and potential gonorrhoea treatment failures, to make sure they are identified and managed promptly."


Monday, 2 November 2015

Citrobacter freundii

Citrobacter freundii
Citrobacter freundii
Citrobacter freundii is a Gram negative rod bacteria that belongs to the Enterobacteriaceae family. They are aerobic and facultative anaerobes. They can cause a number of infections including urinary tract infections and sepsis. According to Whalen et al, 2007, C. freundii represents 29% of all opportunistic infections.
They grow on Chromogenic agar as shown above as pink or purple colonies due to its strong beta-galactosidase activity (Ford, 2010). They are typically citrate positive and can be differentiated from Salmonellae in that they do not decarboxylate lysine (Brooks et al, 1998). They are oxidase negative and catalase positive when tested in the laboratory. They are also negative to indole test which can be used as a rapid test to differentiate it from Escherichia coli which is indole positive from chromogenic agar where they produce similar colour.
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.
Whalen J.G, Mully T.W and English J.C (2007). Spontaneous Citrobacter freundii Infection in an Immunocompetent Patient. Archives of Dermatology 143 (1): 124–125.

Monday, 19 October 2015

Diagnosis of Mycobacterium tuberculosis (TB)

Tuberculosis (TB) is a bacterial infection that causes a major illness and death with approximately 9 million new cases and 1.3 million deaths annually throughout the world caused by members of Mycobacterium tuberculosis complex (MTBC) which includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium pinnipedii, Mycobacterium microti, Mycobacterium caprae and Mycobacterium canettii. However most human cases of TB are caused by the first three organisms in the complex mentioned above. Respiratory infections can also be caused by members of non-tuberculosis mycobacteria called Mycobacterium avium complex (MAC) and includes Mycobacterium avium and Mycobacterium intracellulare.
Mycobacterium tuberculosis is a small, aerobic, nonmotile bacillus that causes TB. TB is primarily a disease of the lung but may spread to virtually any organ of the body or proceed to a generalised infection (miliary tuberculosis) and is characterised by granuloma formation. It contains distinctive cell walls with high concentration of lipids, notably mycolic acids which offers a high degree of protection to the cells and accounts for other properties which include resistance to acids and alkalis, resistance to antibiotics and disinfectants, resistance to drying and osmotic lysis, impermeability to stains and survival within macrophages. It divides every 16 to 20 hours, which is an extremely slow rate compared with other bacteria, which usually divide in less than an hour. General signs and symptoms include fever, chills, night sweats, loss of appetite, weight loss, and fatigue, and significant finger clubbing may also occur. Chest x-ray, tuberculin skin test, acid-fast bacilli stain and culture are the diagnostic methods for TB.
UK standard of Microbiology investigations under the investigation of specimen for Mycobacterium species are a collection of recommended algorithms and procedures covering all stages of the investigative process in Microbiology. These standards are developed under the auspices of the Health Protection Agency (HPA), working in partnership with National Health Services (NHS), Public Health Wales and other professional organisation.
Specimens for the diagnosis of TB and available standard techniques for the detection of Mycobacteria in patient samples
Sputum, Bronchoalveolar lavage (BAL), pleural fluid and tissues samples from any site of the body. Detection of TB includes the initial AAFB staining and microscopy which is vital because results can be available within one hour of receipt of the specimen in the laboratory and several weeks before culture results because of the slow growth of the organism. Therefore, microscopy plays a major role in the patient treatment and management especially in positive cases. However, TB microscopy despite the simplicity and rapid results, lack sensitivity and does not identify drug resistant strains thus if the clinical details suggests TB, then treatment will be given to the patient regardless.
Staining techniques used in TB microscopy include Ziehl-Neelsen (ZN) and auramine stain (AP). Both stains use phenol which acts as a detergent and reduces the hydrophobic effect of the lipids and thus enables the dye to penetrate the cell wall. The stained isolate was then viewed under the fluorescence microscope the bacteria appear brilliant greenish yellow against dark background.
Pre-treatment - Non-purulent liquid specimens are spun down in a centrifuge at 2500 rpm for 10 minutes to concentrate them. The supernatant is then separated into a sterile universal bottle leaving 1ml to resuspend the pellet.
Homogenisation of specimen improves the sensitivity of the culture by permitting the bacteria to be released from the thick sputum and can be achieved by the following methods
(a) Repeatedly vortexing during decontamination process until suspension is fully homogenised.
(b) Treatment with Sputasol (Oxoid Ltd, Basingstoke, UK; containing 100µg/ml dithiothreitol) is used to homogenise the specimen by adding equal volume of 0.1% solution of Sputasol to the sample and vortex intermittently and leave for 15 minutes at room temperature, followed by gentle vortex to assist homogenisation and
(c) Treatment with N-acetyl-L-cysteine (NALC) during decontamination.
Decontamination can be achieved by either by the use of sodium hydroxide (NaOH) or NALC-NaOH. The contaminating normal flora is preferentially killed at this stage (decontamination). Specimens that require decontamination include sputum, bronchial secretions, washings, or biopsies, urines and all other specimens from sites contaminated with normal microbial flora. However, contaminating organisms should not be present in samples obtained by bronchoscopy such as bronchoalveolar lavage (BAL) and any pathogens present will have been diluted by the saline used in bronchoscopy.
(a) 0.7ml of NaOH (0.5N) is added to the specimen and allows to act for 30 minutes at room temperature and vortexing at regular intervals. The specimen is then neutralised with 14ml of sterile 0.067 M phosphate buffer (pH 6.8). Alternatively, follow the above procedure but add 2ml of 1N NaOH (4%w/v) to 2ml of specimen instead of 0.7ml of NaOH (0.5N) and neutralise with 3ml of sterile 0.067 M phosphate buffer (pH 6.8) instead of 14ml.
(b) Add equal volume of working NALC-NaOH solution (2% NALC and 0.5N NaOH, no more than 48 hours old) to the specimen and vortex for approximately 20 seconds. Allow to stand for 30 minutes at room temperature to decontaminate the specimen and dilute the mixture to a minimum of 20 ml with 0.067 M phosphate buffer (pH 6.8). Invert several times to ensure that the content is mixed.
Concentration: Specimens are spun down in a centrifuge at 3000 rpm for 15 minutes to concentrate them. The supernatant is then discarded into a disinfectant leaving 1ml to resuspend the pellet or resuspend in 0.067 M phosphate buffer (pH 6.8).

Specialised techniques available at reference laboratories
TB staining, culture, identification, sensitivity and typing are done at a specialised TB laboratory and the procedures and techniques used are described below:
Culture of Mycobacterium tuberculosis is an important part of the laboratory investigation of TB. Specimens undergo the above pre-treatment processes before culture to eliminate contaminants and concentrate the specimen before culture. There are three types of media used for conventional Mycobacterial culture which includes egg based solid media (Lowenstein-Jensen medium), Agar based solid media (Middlebrook agar) and liquid media.
Lowenstein-Jensen medium (LJ) is the most commonly used media in United Kingdom and is prepared in bottles which are heated while tilted to make slopes. The heat dehydrates the egg proteins so the medium solidifies. Malachite green dye which is inhibitory to most bacteria but not to Mycobacteria is incorporated in the culture medium to prevent the growth of organisms that survived decontamination process. Typical Mycobacterium tuberculosis appears on LJ medium after a couple of weeks of incubation at 35-37 oC as irregular, dry colonies that are beige or buff in colour. The culture is considered negative if no growth after 10-12 weeks of incubation with checks every week for possible acid-fast growth. Presence of Acid-fast Bacilli in positive cultures is confirmed with ZN or AP stain and aliquots are then sent for susceptibility test.
Growth of organisms consumes oxygen and produces carbon dioxide and may be detected by changes in radioactivity, fluorescence, reflectance and pressure. Automated Mycobacterial culture methods such as BACTEC MGIT 960 using the Mycobacterial Growth Index Tube (MGIT) system is based on liquid culture and detects the growth of Mycobacteria faster than conventional culture. Fully automated systems capable of holding up to 960 patient samples continuously (every 60 minutes) monitor the culture bottles and flag new positives cultures usually within 10 – 12 days. It utilises fluorescence technology (O2 reduction). In MGIT, a fluorescent oxygen sensor is embedded in the base of the tube that detects any decrease in O2 dissolved in broth. Oxygen sensor will emit light when exposed to UV with actively respiring organisms consume O2 and reduction in O2 is detected by machine thus the machine flags tube as positive. Positive tubes flagged by machine are then removed, centrifuged for 15 minutes and stained using an AFB stain. Another automated analyser used for Mycobacterial culture is the Biomerieux BacT/ALERT 3D MP which monitors the production and presence of carbon dioxide (CO2) produced by the organism by using a colorimetric sensor and reflected light. As the organisms grow and metabolise substrates in the culture medium, CO2 is produced and is detected by the analyser when the level of CO2 produced reaches a certain threshold. This threshold is determined by the colour change to lighter green or yellow at the bottom of the culture bottle which has an in-built gas permeable sensor. The reflectance units monitored by the analyser increases as a result of the lighter colour and is then recorded every 10 minutes. The colony forming unit (CFU) at the time of detection is approximately 106 – 107 per ml.
Identification of Mycobacterium tuberculosis following isolation is usually done at a National Mycobacterial reference laboratory. It is identified to complex/species level and follows the use of AFB stains (ZN and AP), biochemical, hybridization gene probe and nucleic acid amplification tests (NAATs). Current UK guidelines recommend that a NAATs or hybridization gene probe test which may allow rapid diagnosis of TB should be performed within one working day of isolation of Mycobacterium tuberculosis (National Institute for Health and Care Excellence –NICE and HPA UK Standard for investigation of specimens for Mycobacterium species). NAATs (PCR) analyser used at HPA Freeman Hospital, Newcastle is the Cepheid GeneXpert using the Xpert MTB/RIF cartridge.
Matrix-assisted laser desorption ionisation – time of flight (MALDI-TOF) mass spectroscopy is another automated Mycobacterial identification method and analyses 16s ribosomal proteins and can identify Mycobacterium species within 20 minutes.
MTBC isolates need to be typed which simply means the use of further tests that can discriminate between multiple isolates of the same species. The detection of genomic differences between isolates (genotyping) is the preferred method of typing rather than typing based on the differences in their behaviour (phenotyping).
In UK, the current recommended typing method enables comparisons to be made nationally or internationally. This is known as mycobacteria interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing. It is recommended that an MIRU-VNTR genotype for each new MTBC isolate should be available and entered on the national database within 21 days of mycobacterial reference laboratory receipt for ≥95% isolates.
Diagnosis of latent TB
Diagnosis of latent TB infection involves assessing the host’s cell-mediated immune response by detecting a cytokine called interferon- gamma (IFN-γ). This test does not involve the detection of mycobacteria. Tuberculin skin test or Mantoux test is a screening test for TB used in the detection of latent TB, detection of recent infection and as part of the diagnosis.
The standard Mantoux test in the UK consists of an intradermal injection of two tuberculin units (2TU) of Statens Serum Institute (SSI) tuberculin RT23 in 0.1ml solution for injection and read 48 to 72 hours after administration. A reading is then obtained by measuring and recording the presence or absence of induration. The diameter of the induration which is a hard, dense, raised formation is measured. In the absence of specific clinical details of risk factors for TB, a reading of 6-15mm is more likely to be due to previous BCG vaccination or infection with environmental mycobacteria than TB infection.
Other tests used in diagnosis of latent TB include the Interferon-γ release assays (IGRAs) on a blood sample test called QuantiFERON-TB Gold in-tube. This analysis involves the in vitro stimulation of cells in blood using peptide stimulating antigens (ESAT-6, CFP-10 and TB7.7 (p4)). Enzyme-Linked Immunosorbent Assay (ELISA) detect the production of Interferon-γ is then used to identify responses to these peptide antigens in vitro that are linked to Mycobacterium tuberculosis.

Treatment options for TB
TB can be treated with antibiotics to kill the bacteria. Effective TB treatment is difficult, due to the unusual structure and chemical composition of the mycobacterial cell wall described above, which hinders the entry of drugs and makes many antibiotics ineffective. Antimicrobacterial susceptibility testing can be based on inhibition of growth or detection of generic mutations. Automated liquid systems detect antimicrobial resistance by adding antimicrobial substances to the culture while conventional methods detect resistance by relying on growth, or inhibition of growth of the organism.  The treatment of latent TB usually involves the use of a single antibiotic of either Isoniazid or Rifampicin, while active TB disease is best treated with combinations of a few antibiotics. Latent TB is usually treated to prevent the infection progressing to active state while active TB is treated with a few antibiotics to reduce the risk of the bacteria developing antibiotic resistance. Antibiotic treatment of Mycobacterium tuberculosis includes long term administration (6 months) of multiple antimicrobial agents. Sensitivity testing is performed at the National Mycobacterial reference laboratory. In the UK, the antimicrobial treatment of active TB comprises two stages:
(1) Initial stage of Isoniazid, Rifampicin and Pyrazinamide for two months.
(2) Continuation stage of Isoniazid and Rifampicin for further four months.
Multidrug resistant strains TB (MDR-TB) develops in otherwise treatable TB when the course of antibiotics is interrupted and the levels of drug in the body are insufficient to kill 100 percent of the bacteria. This can happen for a number of reasons such as patients may feel better and halt their antibiotic course, drug supplies may run out or become scarce, patients may forget to take their medication from time to time or patients do not receive effective therapy. Secondly, MDR-TB can become resistant to the major second-line drug groups such as fluoroquinolones and injectable drugs. When MDR-TB is resistant to at least one drug from each group, it's defined as extensively drug-resistant tuberculosis (XDR-TB).
Recent statistics show that approximately 1% of UK isolates of Mycobacterium tuberculosis are MDR with Rifampicin resistance used as a marker for possible MDRTB. MDR are usually resistant to Isoniazid and Rifampicin and are treated with five drugs. The five drugs should be chosen in the following order (based on known sensitivities): Aminoglycoside (such as Amikacin, Kanamycin) or polypeptide antibiotic (such as Capreomycin), Pyrazinamide, Ethambutol, a fluoroquinolone such as Moxifloxacin (Ciprofloxacin should no longer be used), Rifabutin, Cycloserine, a thioamide such as Prothionamide or Ethionamide, 4-aminosalicyclic acid (PAS), a macrolide such as Clarithromycin, Linezolid, high dose INH (if low level resistance), Interferon-γ, Thioridazine and Ampicillin.
Drugs placed nearer the top of the list are more effective and less toxic; drugs placed nearer the bottom of the list are less effective, more toxic, or more difficult to obtain. The recommended treatment of new-onset pulmonary tuberculosis, as of 2010, is six months of a combination of antibiotics containing rifampicin, isoniazid, pyrazinamide and ethambutol for the first two months, and only rifampicin and isoniazid for the last four months and where resistance to isoniazid is high, ethambutol may be added for the last four months as an alternative.

Response to treatment must be obtained by repeated sputum cultures (monthly if possible). Treatment for MDR-TB must be given for a minimum of 18 months and cannot be stopped until the patient has been culture-negative for a minimum of nine months. It is not unusual for patients with MDR-TB to be on treatment for two years or more. Patients with MDR-TB should be isolated in negative-pressure rooms, if possible. Patients with MDR-TB should not be accommodated on the same ward as immunosuppressed patients (HIV-infected patients, or patients on immunosuppressive drugs).

WBCs and RBCs count in sterile body fluids (such as CSF)

The quantity of white blood cells and red blood cells in sterile body fluids (such as CSF) can be obtained by performing a cell count on the uncentrifuged specimen, preferably the last specimen taken, using a modified mirrored Fuchs Rosenthal counting chamber. The cell count of sterile body fluids specimen such as cerebrospinal fluids (CSF) usually gives important clues of presence of infection or not and if present, the infecting organism. Sequential specimens 1 to 4 are usually obtained from one lumbar puncture and specimen 1 is sent to Clinical Biochemistry laboratory while 2 – 4 are sent to Microbiology laboratory. Red cell count is done on the entire 3 Specimen sent to Microbiology laboratory and both RBC and WBC count is performed on specimen 4. Uniform blood staining of all samples suggests previous haemorrhage into the subarachnoid space, whereas reducing counts in sequentially obtained samples suggest bleeding induced by the tap procedure.  CSF obtained more than 12 hours post intra-cranial haemorrhage may show raised WBC counts of up to 500 x 106/l as a result of an inflammatory response.
The procedure for performing a cell count starts with drawing a line with a chinagraph pencil on the external supports of the clean and dry counting chamber and then gently pushes the cover glass onto the counting chamber from the front. The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned. Fill the counting chamber with the specimen using a fine tip sterile pipette and allows to settle for 5 minutes. Observe under x10 objective lens microscope to focus and the x10 or x40 to count cells.
The modified Fuchs Rosenthal counting chamber has nine (9) large triple lined squares; each divided in 16 small squares and has a depth of 0.2 mm. as shown in Figure below.  Each large square is 1 mm2; therefore 5 large triple lined squares are counted to get the count /mm3. The four corner squares and the middle are also counted.  If cells are lying on the triple lines between squares count only the cells lying on the inner two lines of the top and the left side lines.
The ratio of WBC and RBC in a normal blood in WBC1-2: 1000 RBC and under normal conditions the number of white cells in a CSF is <5/mm3 in children and <20/mm3 in adults. In some situations where the sample is turbid or blood stained, with a high expectation of RBC count of >200 mm3, dilution is often performed in sterile saline before loading on the counting chamber. The cell count result must then be multiplied by the dilution factor.

Normal CSF values

Normal CSF values              Neonates                   Adults

Leucocytes                         Neonates              0-30  cells / cu mm   ( x 106/l)
                                           1-4yr old                0-20  cells / cu mm   ( x 106/l)
                                           5yr-puberty            0-10  cells / cu mm   ( x 106/l)
                                           Adults                     0-5   cells / cu mm   ( x 106/l)
Erythrocytes                Newborn                     0-675 cells / cu mm   ( x 106/l)
                                      Adults                        0-10  cells / cu mm   ( x 106/l)

The counting chamber grid