EDITORIAL
Prof. Michael Pepper
MBChB, PhD, MD
NetCare Molecular Medicine Institute, Unitas Hospital

MOLECULAR DETECTION OF PLASMODIUM INFECTION –
A MODERN APPROACH TO MALARIA DIAGNOSIS
Dr. Gerhard Weldhagen
MBChB, MMed (Path), PhD
Molecular Biology Laboratory,
Drs Du Buisson and Partners Inc.,
AMPATH National Laboratory Service

• Malaria can and should still be diagnosed using microscopy and antigen detection methods, where the laboratory expertise exists
• Malaria PCR is highly sensitive and specific, but not cost-effective on a routine basis
• Malaria PCR should be restricted to difficult-to- diagnose clinical cases
• Malaria PCR’s single biggest benefit is the ability to confidently rule out the disease

Microscopical analyses of suitably stained blood smears are still regarded as the “ gold standard” in malaria diagnosis, especially when coupled with highly sensitive immunochromatographic parasite antigen detection methods. Although sensitive,specific and cost-effective, these more traditional laboratory methods may fail, especially during early stages of infection or where prophylactic antimalarials were taken.

The molecular detection of Plasmodium spp. was only feasible once the malaria genome project got underway, mapping complete sequences of several parasite chromosomes. The genome of P. falciparum roughly consists of 22.8 million DNA base pairs; constituting 5279 different genes split over 14 chromosomes. (The human genome by comparison, is a hundred-fold larger, constituting 23 chromosomes and roughly 25 000 known genes.) A remarkable sixty percent of Plasmodium genes are unique to the organism, a very high percentage compared to other eukaryotic organisms, partly suggesting both the evolutionary distance and specialised ecological niche occupied by the species. DNA generally consists of dideoxynucleotide building blocks (known as the purines adenosine (A), and guanosine (G), or the pyrimidines cytosine (C) and thymidine (T)) bound to each other in an elaborate double helix pattern. In normal DNA molecules these bases (A, C, G, and T) occur in fairly equal numbers, making DNA a very stable molecule, able to withstand harsh conditions for very long periods. Plasmodium DNA however, contains large amounts of A and T nucleotides (about 80%), making it unstable and a major stumbling block for molecular biologists to work with. It is this instability that seriously hampered the genome-sequencing project initially, only until laboratory-cloning techniques were perfected, was the malaria-sequencing puzzle solved.

It is with this background that the detection and identification of malaria species by means of the polymerase chain reaction (PCR) came into clinical use. Initial PCR diagnostics consisted of cumbersome processes utilising detection by means of gel electrophoresis, complete with all the inherent problems associated with the procedure, including cross-contamination. The advent of real-time PCR however, changed the whole scenario with concern to malaria diagnostics. Highly sensitive and specific modern real-time PCR platforms obviate the manipulation of amplified DNA products, thereby minimising cross contamination to negligible levels. Several detection chemistries are currently used, ranging from SYBR Green, TaqMan-based probes to hybridisation probes, making accurate malaria species identification on a genetic level, a reality. Real-time PCR detection assays vary in their analytical sensitivity, mainly due to the genetic targets employed in such tests. Reports in published literature provide data suggesting excellent sensitivities, down to a level of 0.2 genome equivalents, which translates to less than one parasite per PCR reaction. Real-time PCR detection of malaria currently employed by our laboratory, exhibit a sensitivity and specificity of 99.5 and 100% respectively, when compared to established methods. The tangible benefit of malaria PCR actually lies in its inherent ability to rule out the disease, making sense out of vague clinical symptoms and confusing laboratory data. With a negative predictive value of 99.6%, this test can truly guide the clinician through problematic clinical cases, adding value to the diagnostic process.

For more information (including references), visit http://www.ampath.co.za
or contact:
Dr Gerhard Weldhagen
Pathologist : Molecular Biology
Tel: 012 427 1777 / 1800
E-mail: weldhageng@ampath.co.zaMolecular  biology laboratory
Tel: 012 427 1833 / 1722 After hours
Microbiologist on call: 012 427 1800

CLINICAL UTILITY OF NT-PROBNP
Dr. Leentjie van Niekerk
MBChB, MMed (Path)
Chemical Pathology Laboratory,
Drs Du Buisson, Bruinette & Kramer Inc.,
AMPATH National Laboratory Service

• NT-proBNP levels, in combination with clinical judgement and other tests, are used for the diagnosis of heart failure (HF) in patients with suggestive symptoms in the acute and out patient setting: levels correlate with the degree of HF

• Can be used as prognostic marker and for risk stratification in HF, acute coronary syndromes and pulmonary embolism

• Could be useful in tailoring therapy in patients with HF

Introduction

B-type (brain) natriuretic peptide (BNP) was first isolated from porcine brain in 1988. BNP is part of the natriuretic family, together with atrial and C-type natriuretic peptides. Pre-pro-BNP is cleaved enzymatically to the prohormone (pro-BNP), which is cleaved to the biologically active BNP and the inactive but more stable amino-terminal portion of the prohormone (NT-proBNP). BNP and NT-proBNP are synthesized and secreted mainly by cardiac ventricular myocytes, released into the circulation in equimolar concentrations, and have similar diagnostic, prognostic and risk stratification capabilities

Pathophysiology

The main stimuli for the acute release of BNP are cardiac wall stretch and tension, in response to volume and pressure overload. Release is also stimulated by ischaemia.

BNP causes vasodilatation, natriuresis, and diuresis, and as such helps to counteract the vasoconstriction and fluid retention triggered by catecholamines, renin and aldosterone, which are increased in patients with heart failure (HF). Other functions include inhibition of endothelin-1, cytokines, ventricular and vascular hypertrophy and remodeling, regulation of coagulation and fibrinolysis, as well as inhibition of platelet activation and platelet coagulation.

Although NT-proBNP and BNP are released into the circulation in equimolar amounts, NT-proBNP levels are as much as 4- to 6-fold higher than BNP because, amongst other things, of differences in clearance mechanisms. Plasma half-life of NT-proBNP is ~120 minutes and it is cleared via the kidneys by glomerular filtration. Intra individual variation in healthy volunteers and patients with stable heart failure is 35%.

Test availablilty and frequency

NT-proBNP is measured in serum (clotted tube) and is available as an emergency investigation. Patients should be tested on admission for HF, before discharge from the hospital and several days to weeks after adjustment of therapy.

Clinical utility of NT-proBNP measurement

1. Diagnostic relevance of NT-proBNP in heart failure.

Heart failure, especially in its early stages, is difficult to diagnose as clinical characteristics are often absent or difficult to interpret. There is therefore a medical need for an objective and reliable test such as NT-proBNP for the identification of patients with ventricular dysfunction and for differentiation of dyspnoea caused by heart failure from pulmonary causes.

The use of NT-proBNP has recently been included in the guidelines of the American and European Societies of Cardiology for the investigation of suspected heart failure. It is best used as a “rule out” test for HF and should not be a replacement for full clinical assessment. Positive results are used to identify patients who need cardiac imaging.

NT-proBNP levels rise in proportion to the severity of heart failure and the New York Heart Association classification of HF. NT-proBNP levels are substantially increased in CHF, often >1000 pg/ ml, compared with minor increases <400 pg/ ml in LV dysfunction without acute CHF.

Guidelines for the use of NT-proBNP in the ACUTE/EMERGENCY setting - algorithm for diagnosing acute congestive heart failure (CHF):

Age-stratified NT-proBNP cut-off points for rule in or rule out of acute CHF in the emergency department (ED):

Guidelines for the use of NT-proBNP in the NON-ACUTE/OUTPATIENT setting - algorithm summarizing recommendations for the diagnosis of heart failure (from NICE Clinical Guideline No. 5 in Natriuretic peptides and the heart: current and future implications for clinical biochemistry. Ann Clin Biochem 42: 432-440, 2005):

Symptomatic heart failure in an out patient setting:RULE OUT: NT-proBNP 125 pg/mL (Sensitivity 96%; Specificity 77%; NPV 97%; PPV 76%)RULE IN: NT-proBNP 500 pg/mL

2. Prognostic relevance of NT-proBNP
2.1. Chronic heart failure

Increased NT-proBNP concentration is associated with increased cardiovascular and all-cause mortality, including sudden cardiac death, independent of age, NYHA class, previous myocardial infarction, and left-ventricular ejection fraction. It is also a predictor of readmission for heart failure and outcome after presentation to the emergency department for heart failure. Among patients admitted with heart failure, NT-proBNP measured prior to discharge is an independent predictor of subsequent mortality or re-hospitalisation for heart failure within the first 180 days after discharge. The risk of these events increases with increasing levels of NT-proBNP (J Am Coll Cardiol 43: 635-641, 2004).

NT-proBNP pg/m/L	Risk of Death or re-admission at 180 days
<1860	15%
1860-3721	60%
>3721	95%

Patients with persistently high levels of NT-proBNP despite aggressive treatment for heart failure are at especially high risk for adverse outcomes.

2.2. Acute coronary syndrome (ACS)

NT-proBNP increases after AMI; the extent of the increase is related to the size of the infarct. It increases monophasic in small infarcts, peaking at 20 hours; and biphasic in larger infarcts with an additional peak at 5 days after admission.

Serial monitoring of patients with ACS showed that a decrease of NT-proBNP within 72 hours was associated with a low short-term cardiac risk (1%-2%) in contrast with an increase or lack of a decrease which was associated with a high cardiac risk (17% -20%). Elevated NT-proBNP identifies patients at risk for adverse left-ventricular remodelling, left-ventricular dysfunction, heart failure, and death, when measured within 1 wk after AMI, with a sensitivity and specificity of 91% and 72% in predicting 2-yr survival. For prediction of death over 24 months of follow-up, an early postinfarction NT-proBNP concentration of 1353 pg/mL had a prognostic accuracy superior to assessment of left ventricular ejection fraction by echocardiography.

In the case of unstable angina, without evidence of myocardial necrosis or heart failure, raised concentrations of NT-proBNP are also associated with an increased risk of death.

The combination of NT-proBNP and either Troponin, CRP or creatinine clearance should provide a better prediction of mortality than either of the markers alone.

2.3. Pulmonary diseases
NT-proBNP increases in acute and/or chronic pulmonary diseases in parallel with the degree of hypoxia and right heart overload. Increased NT-proBNP concentration can be used as a predictor of a major adverse cardiovascular event following pulmonary embolism.

2.4. Renal disease
Optimal cut-off values for the diagnosis of CHF in renal disease need to be established, with advanced stages of renal failure requiring higher cutoff values.

2.5. Diabetes
NT-proBNP levels predict cardiovascular morbidity and mortality in patients with diabetes.

2.6. General population
The role of NT-proBNP screening in the general population for asymptomatic LV dysfunction is not recommended. However, by targeting high risk individuals the accuracy of NT-proBNP as a screening tool can be improved. One study demonstrated that NT-proBNP could independently predict risk of death, heart failure, atrial fibrillation, and stroke over a mean follow-up period of about five years in a cohort of 85-year-old individuals from the general population.

3. Effect of treatment on NT-proBNP levels
Monitoring of NT-proBNP may be useful in guiding therapy. NT-proBNP-guided treatment of HF reduced total cardiovascular events and delayed time to first event compared with intensive clinically guided treatment. Cardiovascular death, admission, and new episodes of decompensated HF were all lower in the NT-proBNP group <1680 pg/mL.

Whereas NT-proBNP levels decrease with most antihypertensives (see table), the effects of β blockers are more complex. Because adrenergic stimulation inhibits release of natriuretic peptides, initiation of β blockade will slightly increase natriuretic peptide concentrations. However in the long-term, addition of β blockers results in improvements in haemodynamic variables and left ventricular function, with a net effect of reduction in NT-proBNP levels.

NT-proBNP could ultimately prove useful in helping doctors to select the appropriate drugs and drug doses, and of the need for more invasive, non-pharmacological strategies such as implantable defibrillators, ventricular assist devices, or cardiac transplantation. NT-proBNP levels measured after stabilization on treatment were more significant predictors of death and further events than baseline values.

Conclusion

The NT-proBNP assay has provided us with a stable, objective, sensitive and specific marker for cardiac function, with levels correlating with the severity of cardiac dysfunction. It has been included in the diagnostic work-up for HF, and is best used as a RULE OUT marker for exclusion of patients without HF. It is also useful as prognostic and risk stratification marker in numerous disorders, as well as for tailoring therapy in HF. Specific cut-off points still need to be established, especially in patients with renal failure.

For more information (including references),
visit http://www.ampath.co.za  or contact:

Dr Leentjie van Niekerk
Pathologist:
Chemical Pathology
Tel: 012 427 1858 / 1800
E-mail: leentjie@dubuisson.co.za

Chemical pathology laboratory
Tel: 012 427 1852 / 1853After hours
Chemical Pathologist on call: 012 427 1800