Discipline - Haematology
Basic cerebrospinal fluid (CSF) Profile (CSF leukocyte count, CSF differential leukocyte count and CSF protein and glucose)
Haemocytometer / automated haematology analysers with body fluid mode
First added in 2020
Aid to diagnosis
CSF leukocyte count: To aid in the diagnosis of bacterial, mycobacterial, fungal and viral meningitis
Summary of evidence evaluation
No systematic reviews of relevant evidence for these tests are available, and there is little direct evidence for their accuracy (relating to disease) and impact. The basic CSF profile is part of standard practice in evaluating patients suspected of having meningitis. The tests are done as an early part of a strategy and may be used to decide initial therapy. There is strong evidence that early treatment affects outcomes and the case for early diagnosis is strongly made, although it is not clear how the CSF profile affects timing of treatment. There is some evidence (one study) that the CSF profile can help give a first indication of the most likely cause, but accuracy is not high. Further microbiological testing (gram stain and culture) is essential to obtain definitive diagnoses. The full evidence review for this test category is available online at: https://www.who.int/medical_devices/diagnostics/selection_in-vitro/selection_in-vitro-meetings/new-prod-categories_3
Summary of SAGE IVD deliberations
CNS infections are life-threatening conditions of public health importance that require rapid and accurate diagnosis and early treatment to decrease morbidity, mortality and late sequelae. There may be a lack of prospective studies on the diagnostic accuracy of using the basic CSF profile in CNS infections; but the test has long been embedded in clinical practice and is included in almost every relevant guideline as an aid to diagnosis. There are three relevant tests: CSF leukocyte count, CSF differential count, and CSF protein and glucose. Each one uses a different assay format; but in each case the test is usually performed as the first step in a sequence of tests and cannot provide a definitive diagnosis on its own. Nevertheless, SAGE IVD members confirmed that the test has a clear role in clinical practice as there is no other test available for acute meningitis and it provides an immediate result that can influence patient management decisions. The CSF tests themselves are fairly routine and can be done by hospitals with laboratory facilities. But SAGE IVD did raise some concerns about their use, particularly around variation in test results. Manual methods using haemocytometers can have high inter-observer variation and require a high level of training. Automated analysers are more expensive and tend to vary in terms of the technology platform used for cell counts. Not all of them have regulatory approval for the body fluid mode; and some have been documented to have poor precision at low CSF counts. SAGE IVD noted that the CSF profile is essentially a specimen type and that it can be used in many different conditions. The group emphasized the need to clarify that in this case it is being considered specifically as an investigation tool for meningitis.
SAGE IVD recommendation
SAGE IVD recommended including the CSF profile (leukocyte count, differential leukocyte count, protein and glucose) test category in the third EDL: • as a disease-specific IVD for use in clinical laboratories (EDL 3, Section II.a Haematology,); • using a haemocytometer or automated haematology analyser with body fluid mode (CSF leukocyte count), Wright–Giemsa-stained smears or automated haematology analyser with body fluid mode (CSF differential leukocyte count), automated or semi-automated chemistry analyser (CSF protein and glucose); • to aid in the diagnosis of bacterial, mycobacterial, fungal and viral meningitis. SAGE IVD requested the addition of a note to the test category entry in the EDL stating that definitive diagnosis requires microbiological confirmation, including through gram staining, antigen testing, nucleic acid testing and culture. The group further recommended reviewing all entries in EDL 2 and adding CSF as a specimen type as and where relevant in the next edition of the EDL (EDL 4).
Details of submission from 2020
Disease condition and impact on patients Infectious neurologic disease remains a significant threat to public health, particularly in resource-limited settings. Globally, there are an estimated 2.8 million cases each year, many in sub-Saharan Africa (1). Nearly half of these are bacterial meningitis (1.2 million cases). Moreover, although the number of viral meningitis cases has significantly decreased over the past decade, this is also significant at approximately 400 000 annual cases. The prevalence of fungal meningitis often correlates with the prevalence of immunocompromised populations. For example, a recent study of 5500 lumbar punctures in South Africa showed 63% (n 514) of the abnormal studies were attributable to cryptococcal meningitis (2). Other data suggest there are approximately 1 million cases of cryptococcal meningitis yearly among persons living with HIV, with more than 180 000 deaths, 75% of which occur in sub-Saharan Africa (3). All these diseases can have enduring impacts on patients. For example, patients who recover from bacterial meningitis often suffer long-term cognitive deficit, bilateral hearing loss, motor deficit, seizures, visual impairment and hydrocephalus. Less severe problems include behavioural disorders, learning difficulties, unilateral hearing loss, hypotonia and diplopia. Similarly, most patients who recover from mycobacterial meningitis are left with cognitive impairment, motor deficits, optic atrophy and cranial nerve palsies (4). Does the test meet a medical need? The basic CSF profile – evaluation of CSF WBC count, RBC count, protein and glucose – plays an integral role in diagnosing and managing a broad range of infectious and non-infectious diseases of the central and peripheral nervous system. The test is required to diagnose, and aid in the diagnosis of, bacterial, mycobacterial, fungal and viral meningitis with the help of CSF gram stain, microscopy, polymerase chain reaction (PCR)-based testing and culture. Incorporating the basic CSF profile into the EDL not only will enable more rapid, accurate and effective management of individual patients with suspected meningoencephalitis but also has the potential to make a major impact on public health internationally. Specifically, it could reduce antimicrobial resistance caused by antibiotic misuse, improve public health monitoring and reduce overall health care costs related to patients with central nervous system (CNS) infections. How the test is used Although no single measure is diagnostic, the basic CSF profile (RBCs, WBCs, glucose, protein) in addition to CSF gram stain is used to determine the initial likelihood of bacterial, fungal or viral meningitis and so guide the decision to begin or continue antibiotic therapy. A combination of pleocytosis, decreased glucose and elevated protein suggest a bacterial etiology for meningitis. Lymphocytic pleocytosis, normal glucose and elevated protein suggest a viral etiology but may also reflect fungal and mycobacterial meningitis. The results of the CSF profile are therefore not confirmatory in isolation. Several organizational guidelines include algorithms of varying detail for diagnosing and treating meningitis: these begin with evaluating for contraindications to lumbar puncture, followed by lumbar puncture and/or empiric antibiotics. Results of the basic CSF profile and gram staining consistent with bacterial meningitis indicate continued antibiotic use. Other confirmatory testing, including latex agglutination assays, PCR-based tests and CSF culture, can be used to confirm specific viral and bacterial etiologies. In children, a meningitis score has been validated to help predict bacterial meningitis (5). This score includes five components: positive CSF gram stain, CSF absolute neutrophil count (> 1000 cells/L), CSF protein (> 80 mg/L), serum WBC (> 10 000 cells/L) and presence of seizures. The presence of one or more of these components demonstrates a 99.8% sensitivity for bacterial meningitis. The score is not yet used in paediatric guidelines, but it reiterates the importance of the basic CSF profile in predicting bacterial meningitis among children. Mycobacterial and fungal etiologies require other confirmatory laboratory testing. For cryptococcal meningitis, cryptococcal antigen testing in serum and/or CSF is recommended as well as specialized microscopy using India ink staining to confirm diagnosis. Diagnostic challenges exist around mycobacterial meningitis, but the first step remains the basic CSF profile analysis, followed by acid-fast bacterial culture (Ziehl–Neelsen) and imaging and tissue pathology where possible.
Public health relevance
Prevalence and socioeconomic impact In the most recent global burden of disease (GBD) study, neurologic disease is the number one group cause of DALYs, causing 12% of global DALYs (1). Overall, meningitis contributed 8% of the neurologic-disease DALYs, although this figure varies across income categories and age ranges. For, example, meningitis ranks fourth overall but second in sub-Saharan Africa. Among children 5 years old and younger, meningitis is the primary cause of neurological DALYs, contributing at least 25% of neurologic years of life lost from birth to 25 years of age. Meningitis also causes deaths. All-cause, global mortality attributed to meningitis approaches 320 000 deaths per year. Importantly, mortality estimates more than double in resource-limited settings compared with resource-rich countries (1). Bacterial meningitis affects 1.2 million people each year (6). In the 2016 GBD study, bacterial meningitis caused by Haemophilus influenzae and meningococcus accounted for 600 cases of meningitis, approximately 200 deaths and 20 years lived with disability per 100 000 in the world (1). The GBD study identified bacterial meningitis as the 27th most burdensome condition; but a sub-analysis of rural Burkina Faso ranked it sixth (7). Around half (54%) of all cases and 46% of meningitis-associated deaths occur in children under 5 years of age (1). Mortality is 50% in LMICs compared with 20% in high-income settings (8). This is partly because H. influenzae, which has a relatively high mortality rate, is rarely seen in regions with good vaccination programmes. The risk of sequelae similarly varies across income settings, being almost double among Africans compared with Europeans (9). In terms of specific organisms, major sequelae occur in 25% of patients with pneumococcal infection, 10% with H. influenzae and 8% with meningococcal infections. Viral meningitis similarly varies across income settings. A prospective registry of suspect meningitis in the United Kingdom found that a third of adult patients had viral meningitis, with 55% caused by enteroviruses, 24% herpes simplex virus (HSV), and 19% varicella (10). Data on HSV type 1 CNS infections in Africa indicates that the condition overall is more common and virulent than in the industrialized world (11). But even in relatively well-resourced settings, the mortality of HSV infections remains high: a recent report across 47 intensive care units (ICUs) in France found a 17% mortality rate among patients with HSV encephalitis, with 54% of survivors having significant sequelae with a modified Rankin score of ≤ 2.6 (12). Mycobacterial meningitis includes CNS tuberculosis (TB), which is the most severe manifestation of TB and accounts for around 1% of all TB cases and 5–10% of all extrapulmonary TB cases. An estimated 100 000 cases of extrapulmonary TB involving CNS occur globally each year. Among HIV-negative individuals, CNS TB is associated with a 20–55% mortality rate, with more advanced disease at the time of diagnosis resulting in higher mortality. For those with HIV infection, mortality is higher at 40–75%. Among survivors of CNS TB, delayed diagnosis contributes to the development of hydrocephalus and infarctions, particularly in the basal ganglia and internal capsule. A US-based study identified sequelae in 54% of survivors, including stroke in 15% and epilepsy in 12% (13). Studies from Mexico have shown that clinical outcomes are improved for patients with a bacteriologically confirmed diagnosis (14). Fungal meningitis is relatively rare and is caused by CNS fungal infections. People with weakened immune systems are at higher risk of getting fungal meningitis, and recovery is possible only with timely diagnosis and treatment; definitive diagnosis requires CSF analyses or biopsy. Delayed diagnoses increase the risk of stroke, hydrocephalus and death. The annual incidence of CNS fungal infections has been rising as the number of immunocompromised people continues to grow, driven first by the HIV epidemic and then by the increase in noncommunicable diseases, including diabetes. The greatest burden of CNS fungal infections is in HIV-infected individuals, among whom cryptococcal meningitis remains one of the most common causes of death, with a mortality rate of 15–39% (15, 16). Among survivors of CNS fungal infections, strokes that occurred during the acute infection or during delayed diagnosis contribute substantially to long-term morbidity (15).
WHO or other clinical guidelines relevant to the test
Many national and international guidelines incorporate the basic CSF profile into the diagnosis and management of patients with diseases of the central and peripheral nervous system, and even certain systemic diseases. Examples include guidelines from the IDSA and the American Society for Microbiology (17–19), the European Federation of Neurological Societies (20, 21), the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (22), and the British Infection Society (23, 24). In patients with suspected acute bacterial meningitis, practice guidelines recommend or strongly recommend a lumbar puncture as soon as safely possible, a CSF basic profile, and a CSF gram stain or culture (level of evidence: I; grade of recommendation: A) (18–23). Guideline consensus for typical CSF profiles in acute bacterial meningitis includes a WBC count of > 1000 cells/mm3 (ranging from 1 to 10 000, with neutrophilic predominance), decreased CSF glucose with a CSF : serum glucose ratio of less than 0.4–0.6, and elevated CSF protein (18–20). Guideline consensus for typical CSF profiles in viral meningitis includes a WBC count of 5–1000 cells/mm3, low to normal glucose with a CSF : serum ratio of > 0.4–0.5 and normal to slightly elevated protein. A normal CSF profile can be used to rule out infectious causes of meningoencephalitis, though this can be seen in rare circumstances, for example in immunocompromised patients and neonates (8, 10). In the case of bacterial meningitis, it is recommended that CSF gram stain and culture (included in the current EDL) be used to help identify specific causative bacterial organisms to help tailor the antibiotic regimen and duration of steroid therapy. Similarly, practice guidelines recommend CSF analysis to diagnose and treat CNS TB when there is high clinical suspicion based on history, examination or imaging (23). While not the gold standard for diagnosis, the basic CSF profile analysis plays an important part in guiding treatment decisions for CNS TB, as the diagnostic yield of CSF microscopy or culture is often low, requires large volumes of CSF and may take days to weeks to return positive. Guidelines outline expected findings of CSF leukocytosis (lymphocytic predominance, which can be falsely low in HIV-infected patients), elevated protein and a CSF : serum glucose ratio of less than 0.5 (23). Finally, clinical practice guidelines recommend getting a CSF basic profile and CSF culture or gram stain when clinically concerned for health care-associated causes of ventriculitis or shunt infections and CNS abscesses (19).
Evidence for diagnostic accuracy
No systematic reviews were available at the time of this review. Khatib et al. (25) describe the accuracy of manual analysis of CSF in meningitis. Among a number of clinical and laboratory markers, CSF pleocytosis best differentiated between bacterial meningitis and other etiologies (area under curve > 0.95). A total of 48 published studies were available comparing manual and automated methods or evaluating automated methods for cellular and biochemical analysis of CSF. Studies comparing manual and automated methods found that semi-automated or fully automated methods: • are comparable to manual methods for cell count analysis (RBCs and nucleated cells); • have good agreement with manual methods (kappa of 0.8 or above, and most studies report r2 of > 0.98) (26–28); • have good inter-assay agreement; and • save time and costs (29). Studies noted, however, that diagnostic agreement varies with different cell counts (27). At low nucleated cell/WBC counts, the agreement was low (< 20 cells/µL in most studies) (30, 31). Furthermore, at low or abnormal differential counts, manual review of cell counts should follow automated counts (32–34). Recent updates in analyser technology and software may have improved detection at lower counts (35, 36). But a 2019 study by Zelazowska-Rutkowska et al. (37) in children reiterates the need for manual review of all abnormal counts reported on automated analysers. An initial 1980 study by Warren et al. (38) evaluated automated analyser thresholds for CSF glucose and protein and reported very high thresholds. In 2017, Lefrere et al. (39) reported CSF protein assessment on analysers with a kappa of 0.93 with index methods; glucose testing also correlates well with reference methods (r2 of > 0.98). Londeree et al. (40) evaluated the POC utility of two biochemical analysers for CSF protein and glucose and concluded that this use correlated well with in-hospital use.
Evidence for clinical usefulness and impact
The basic CSF profile remains the reference standard for initial management of meningitis. Its established diagnostic accuracy allows for immediate decisions regarding antibiotic and antiviral treatment. Consequently, studies focusing on clinical utility are futile, as obtaining a basic CSF profile is established standard practice. Some data do, however, exist regarding the impact of delayed analysis and further research is exploring the prognosis potential of the CSF basic profile after the diagnosis is made. Evidence of prognostic value A 2004 nationwide analysis of meningitis patients (696 episodes) in Dutch centres by van de Beek et al. (41) found low CSF WBC count to be an independent predictor of poor outcome. Auburtin et al. (42) published a multicentre analysis also indicating the prognostic value of CSF cell counts in meningitis (pleocytosis of > 1000/mL correlated with poor outcomes). Julian-Jimenez et al. (43) performed a non-systematic review of data from 59 articles and concluded that CSF pleocytosis and low glucose are independent predictors of poor outcome in meningitis. Evidence of impact in antibiotic administration delay: Michael et al. (44) performed a single-centre analysis of 92 patient records and concluded that delay in lumbar puncture (and therefore subsequent CSF analysis) delays the administration of antibiotics. Proulx et al. (45) similarly concluded from a single-centre retrospective study that diagnostic-treatment algorithms (and, therefore, delay in performing lumbar puncture and CSF analysis) impact antibiotic treatment and mortality.
Evidence for economic impact and/or cost–effectiveness
The cost for basic CSF profile varies globally, as resource expenditures change with different health systems, cultures and sociopolitical contexts. Though there are limited global data, estimates suggest a complete profile in sub-Saharan Africa costs as little as US$ 9 (46). As an approximate estimate for monetary cost, the Centers for Medicare and Medicaid Services in the USA cites the cost of the total basic CSF profile at approximately US$ 28.74, with individual components costing: US$ 5.25 for each RBC and WBC count; US$ 4.37 for glucose in body fluid other than blood; and US$ 19.12 for CSF protein assay (47). But private laboratories charge approximately US$ 109.50 for the full CSF profile in the USA (48). Documented costs do not specify whether costs reflect manual or automatic cell counting. The most cost-effective approach to caring for CNS infections is vaccination against threatening organisms such as H. influenzae (49, 50). Most cost analyses on CNS infections do not provide specifics in terms of pathogen but address meningoencephalitis overall. One exception is a 2006 US-based study, which showed that delayed lumbar puncture was a strong predictor of higher cost of care for meningitis (51). In a 2016 study in China, the incremental cost–effectiveness ratio of the Hib vaccine compared with no vaccination was US$ 13 640 at market price, which was less than three times the GDP per capita of China (52). Data on the cost–effectiveness of manual microscopy compared with automated microscopy for assessing CSF cell count are sparse, possibly because manual microscopy remains the standard of care and there is limited use of automated analysers for CSF cell count worldwide. Still, existing data suggest that while automated methods require larger initial investment, the decreased labour and test timing make a quick return). A study in the International Journal of Laboratory Hematology (53) compared the Fuchs–Rosenthal manual counting method with the Sysmex XE-5000 and found that the average time for each manual cell count was 635 seconds, which was significantly greater than the 85 seconds required for the automated method. The same study reported that the total analytical performance cost (including personnel costs, material expenses, laboratory equipment) for the counting chamber was €6.74 for the manual counting method compared with €1.22 for the XE-5000 analyser (53). A study from sub-Saharan Africa shows that diagnostic algorithms that start with the basic CSF profile to determine the need for further testing can provide more efficient care through better antibiotic stewardship and less expensive upfront testing (47).
Ethical issues, equity and human rights issues
Laboratory capabilities vary widely, depending on country and location; quality of laboratory facilities and clinical care also varies between high- and low-income settings. While there is an ethical issue with the variability of hospital-based infrastructure, the proposed CSF test itself does not pose significant ethical challenges. There are no current competing interests attached to the use of this test, and if any arise these shall be discussed openly and officially declared. The use of this test is not sponsored by any pharmaceutical company and shall not be to the financial benefit of a sponsor. This test shall not in any way increase risk of loss of confidentiality, and the samples will be handled using appropriate procedures in the management of human samples as per requirements. CSF analysis is an essential diagnostic laboratory test that is low cost and fundamental to the diagnosis and treatment of neurological illnesses, including infectious diseases and non-infectious disorders.
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