Medical Notebook

P. falciparum:

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Typical incubation time: 7 to 30 days.

If a person is taking chloroquine and if the parasite is partially resistant, there may be temporary suppression of a malaria attack. The fever is generally irregular. If the attack is not treated, after a few weeks a regular fever pattern will develop with peaks every 2 days (tertian malaria, so called because the fever reappears on the third day). This is rare in everyday clinical practice, however. At the beginning of the attack the symptoms are similar to influenza: general malaise, tiredness, muscle pain, headache but in general without respiratory tract problems or runny nose. These symptoms are not very specific. After a while the muscle pain and headache become worse. Sometimes there is also abdominal pain and diarrhoea. Rarely there is a classic attack: this lasts for approximately 12 hours and occurs every 48 hours. At first cold shivers with high fever occur, followed by an intense feeling of heat and fever, leading to a sweating stage with a drop in fever. Most falciparum attacks do not follow this classic pattern, however. So what is referred to as a classic attack is paradoxically not the general rule.

P. vivax and P. ovale:

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The incubation time is a few weeks to years. The awakening of dormant parasites in the liver (hypnozoites) explains late relapses. The fever is sometimes regular (every 48 hours), especially in cases of recrudescence (tertian malaria). In 1922 P. vivax was introduced for the treatment of neurosyphilis. The bacterium which causes syphilis has little resistance to heat, it was hoped that the high fever would kill the bacteria (Treponema pallidum).

P. malariae:

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The incubation time is 3 weeks to many years. The very late attacks are probably not due to awakened hypnozoites (to date these have never been detected) but due to the activation of blood parasites which are present at a very low concentration. Fever peaks may occur every 72 hours (quartan malaria).

Malaria: Mixed infections

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Mixed infections do occur, but for reasons which are unclear they are much less common than would be expected based on the prevalence of the individual species. Under-reporting may play a part, but this is probably a real phenomenon (partial cross-immunity to heterologous species?, biological interference?).

Clinical picture, the natural course of malaria

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Children are very susceptible to infection. The highest mortality is found in children below the age of 5 years. Gradually, after repeated infections, a partial immunity develops in those who survive. There is a high degree of tolerance to the infection in adults, provided that they live in a stable malaria region. This semi-immunity (premunition) is maintained by repeated infections and mild latent infections. It disappears after approximately 6 to 24 months if there is no further infection (e.g. a stay in a non-malaria region). This partial immunity is reduced during pregnancy. A pregnant woman is at increased risk of hypoglycaemia and cerebral malaria. Malaria is an important cause of severe (sometimes spectacular) anaemia in the mother, low birth weight, premature birth, abortion and increased perinatal death. Chondroitin sulphate and hyaluronic acid, both present in abundance around the syncytiotrophoblasts of the placenta, are mucopolysaccharides (glycosamine glycanes) which act as receptors for red blood cells infected with P. falciparum. Infected cells accumulate in the placenta, resulting in reduced placental function. Probably there are also other receptor molecules. The placental barrier is very seldom passed. Congenital malaria is not common and occurs chiefly in neonates of non-immune women. Neonates of semi-immune women receive transplacental anti-Plasmodium antibodies. Due to this passive resistance in the first 3-6 months they are at a lower risk of malaria. AIDS has no direct influence on malaria, although if routine blood transfusions are given for severe anaemia, there is a risk of infection by this route.

Several observations of humans infected with both malaria and helminths suggest that co-infection provides a benefit to either parasite. The evidence indicates that malaria patients co-infected with helminths are protected from severe malaria, possibly through skewering of the immune response towards T helper (Th)2 immunity.

Clinical picture, acute severe malaria

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Acute severe falciparum malaria is a medical emergency. This encompasses:

Coma (the patient cannot be woken)
Repeated generalised convulsions
Hypoglycaemia: reduced consciousness, aggressive behaviour
Severe anaemia: weakness, polypnoea, pale mucosae
Tendency to spontaneous bleeding (pronounced thrombocytopaenia)
Circulatory collapse (shock); cf. algid malaria
Pulmonary oedema (dyspnoea and bilateral crepitations) ± ARDS
Haemoglobinuria (dark urine)
Kidney failure: the urinary flow should be monitored and kept above 400 ml/24h.
Acidosis (chiefly due to lactic acid): rapid deep respiration. If too many salicylates are given, this may exacerbate the acidosis (not unusual in febrile patients).

Other important signs are: confusion without coma, extreme generalised weakness, jaundice, very high fever (hyperpyrexia). The priorities are cerebral involvement, severe anaemia, hypoglycaemia and kidney failure, and the presence of hyperparasitaemia. The degree of parasitaemia correlates with the severity of the symptoms: the higher the parasitaemia, the greater the risk of severe symptoms. It should be borne in mind that the parasitaemia (the percentage of parasitised cells that are found in a smear preparation) changes by the hour. This is because the red blood cells with mature P. falciparum parasites (schizonts) attach themselves to the small capillaries of deep organs, and are not found in a thin blood smear. A parasitaemia of 0.5% is already severe, 2% is pronounced, and patients with a parasitaemia of more than 10% have a relatively poor prognosis. Over 25% is often fatal. Another consideration is that a parasitaemia of 3% in someone who still has a normal red blood cell count, is different from a parasitaemia of 3% in an anaemic patient.

Malaria: Hypoglycaemia

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Hypoglycaemia may quickly lead to general deterioration and coma. It is common in children (up to 25%) and pregnant women. Glucose may be life-saving. If the glycogen store in the liver is low (malnutrition) the risk of hypoglycaemia increases [glycogen is converted to glucose = blood sugar]. The conversion of glycogen to glucose is also inhibited by certain cytokines which are released during infection with P. falciparum. [Hypoglycaemic effects of TNF-α and possibly interleukin-1 and TNF-β]. The parasites themselves also use glucose for their metabolism and contribute to the hypoglycaemia if they are present in large numbers. Quinine can stimulate the secretion of insulin from the pancreas and in this way can also contribute to hypoglycaemia.

Algid malaria

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The term “algid malaria” (L. “algidus" = cold) is obsolete. The condition is characterised by hypotension with progression to shock. The patient is clammy and often feels cold. There is no fever. Often there is septicaemia with Gram-negative bacteria. Mortality is high. As well as therapy with quinine, treatment with antibiotics and IV fluid administration is of great importance. Shock seldom occurs in malaria if there is no septicaemia. Splenic rupture can also cause hypovolaemic shock, however.

Malaria: Splenic rupture.

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Splenic rupture. This may occur spontaneously or after an unobserved trauma. This complication can occur in P. falciparum, P. vivax, P. ovale or P. malariae. The presence of intraperitoneal fluid is suggestive in this context. In these cases ultrasound can often detect a splenic haematoma, splenic rupture or intraperitoneal fluid. A diagnostic peritoneal lavage may be indicated.

Cerebral malaria

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Cerebral malaria is the main cause of death (80 %) in falciparum malaria. This complication occurs chiefly in non-immune persons (children, travellers). Cerebral signs include confused behaviour, psychosis, convulsions, stupor, coma, paralysis. Unlike meningitis, there is no real neck stiffness (pain) or photophobia (intolerance to light) but neck retraction and opisthotonos may occur. Sometimes the difference between neck stiffness and neck retraction is not clinically clear. It is typical of the coma that it develops swiftly in 75% of cases and also quickly disappears. If a child survives cerebral malaria it has approximately a 10% chance of significant sequelae. Children with cerebral malaria and with a normal eye fundus have a good prognosis, while papiloedema and retinal bleeding suggest a guarded prognosis. Repeated generalised convulsions should not be regarded as "normal" febrile convulsions. Severe convulsions with contraction of the abdominal muscles and compression of the stomach, may cause reflux of gastric acid and food into the pharynx. Aspiration of gastric contents into the lungs is a real danger as this may result in Mendelson’s syndrome or aspiration pneumonia. IM phenobarbital is sometimes given as a prophylactic measure (a dose of 3.5 mg/kg up to 10 mg/kg). If there are convulsions, these are stopped by administering diazepam (Valium®) IV or paraldehyde IM. Paraldehyde should be drawn up into a glass syringe (not plastic). A CT scan or MRI scan of the brain of patients with cerebral malaria shows few abnormalities except fpossibly an increased cerebral volume. Herniation of the brain stem is a rare event.

If confronted by a febrile coma or confusion with fever in the tropics, glucose must be administered (preferably IV), quinine therapy should be instituted and a lumbar puncture carried out without hesitation (to rule out meningitis). Of the persons who will die in hospital due to cerebral malaria, 50% of the fatalities occur within the first 12 hours after admission. At autopsy countless petechiae can be seen in the brain. Small ring-shaped haemorrhages also occur around cerebral blood vessels.

Malaria: Severe anaemia

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Severe anaemia occurs due to haemolysis (of both parasitised and non-parasitised red blood cells – the latter via immune-mediated mechanisms), due to excessive action of the spleen i.e. hypersplenism (until weeks after the infection), due to possible haemorrhages (low blood platelets, splenic rupture) and due to disturbed production of new blood cells in the bone marrow (dyserythropoiesis), including that due to TNF-α. Malaria pigment interferes with the differentiation of blood cells in the bone marrow and can contribute to the anaemia.

Malaria: Hyperpyrexia

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Hyperpyrexia should be treated by cooling the patient and administering paracetamol. It is assumed that malaria fever is caused when lysis of the red blood cells releases malaria pigment (haemozoin) which is absorbed by the reticulo-endothelial system. This in turn releases endogenous pyrogens (cytokine network). The concentration of tumour necrosis factor in the peripheral blood correlates with the severity of the malaria. In cases of repeated malaria attacks the liver, spleen and bone marrow are stained black by the enormous amounts of haemozoin.

Malaria: Febrile convulsions

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Febrile convulsions are generalised tonic-clonic convulsions. They only occur in children between the age of 6 months and 5 years and will not be repeated during the same fever episode. They occur during the phase in which the fever is rising fast. They always last less than 15 minutes (including postictal coma) and there is never postictal hemiparesis. It is important to differentiate between febrile convulsions and convulsions during fever (e.g. cerebral malaria, meningitis, cerebral abscess). Approximately 2% of children have a tendency (possibly genetic) for febrile convulsions. The risk that epilepsy will develop in this group of patients is no greater than in children without febrile convulsions. Brief and sporadic attacks have a good prognosis. No maintenance therapy with anti-epileptic agents must be instituted in cases of febrile convulsions.

Black water fever

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Black water fever is a severe but life-threatening complication. Acute massive haemolysis occurs. It has been observed after taking halofantrine, artemisinin-derivatives and after irregular use of quinine. The precise mechanism is not known. The parasitaemia is generally very low. There is high fever, jaundice, back pain, shock and very dark urine. Renal insufficiency occurs: the urine production is very low (oliguria) or zero (anuria). Mortality is very high. When quinine was no longer used prophylactically, black water fever became very rare. As this product is increasingly back into use, it can be assumed that this complication will again become more common. Differential diagnosis should be made with leptospirosis and viral haemorrhagic fever. Acute renal failure may also be caused by shock, hypovolaemia with reduced renal circulation, DIC (diffuse intravascular coagulation), obstruction of the renal glomeruli by parasitised red blood cells and by the precipitation of released haemoglobin in the kidney (pigment nephropathy). The combination of these factors can result in acute tubular necrosis. Glomerulonephritis may occur in chronic quartan malaria, but this complication plays no part in acute renal problems.

Pulmonary oedema is a common complication of severe malaria

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The dividing line between overhydration and dehydration is narrow. Adults easily develop non-cardiogenic pulmonary oedema if there is limited fluid overload, but on the other hand dehydration and hypovolaemia may lead to hypotension, shock and renal failure. As a guideline the central venous pressure should be kept around 5 cm H₂O. If intensive invasive monitoring is available (e.g. Swan-Ganz catheter in an intensive care unit) an attempt should be made to keep the pulmonary capillary wedge pressure around 15 mm Hg. [The PCWP reflects the pressure in the left atrium]. Pneumonia is observed quite often if coma lasts for longer than 3 days. ARDS (acute respiratory distress syndrome) may occur. This is caused by diffuse damage to the vascular endothelium and the alveolar epithelium. There is a rapid progression towards dyspnoea, arterial hypoxia, bilateral patchy pulmonary infiltrates due to pulmonary oedema with a protein-rich fluid. The treatment is both aetiological and symptomatic: artificial ventilation, with or without intubation or an endotracheal cannula, possibly with NO, high-dosed oxygen and positive end-expiratory pressure (PEEP). Surfactant administered via aerosol might be helpful in this situation, although it is often not available. Further data is needed.

Clinical picture, chronic falciparum malaria

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Where P. falciparum is partially resistant to chloroquine, the parasite may be suppressed, but will remain present. This may lead to a whole range of clinical pictures, from asymptomatic parasitaemia through to mild aspecific symptoms, to significant chronic malaise and fatigue. Curative therapy with Malarone®, for example, produces rapid improvement.

Clinical picture, hyperreactive malaria splenomegaly (HMS)

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Some adults have a very strong immunological reaction to P. falciparum infection. The level of IgM in the blood is very high. Due to the polyclonal immune stimulation, all kinds of auto-antibodies can appear. Immune complexes are formed, and are removed by the reticulo-endothelial system, which leads to splenomegaly and sometimes enlarged liver. In these individuals the spleen swells and also breaks down normal, unparasitised blood cells. The number of parasites is very low, but very high concentrations of anti-Plasmodium falciparum antibodies can be detected. The splenomegaly disappears after curative therapy with, e.g. quinine + tetracyclines followed by months or even years of adequate malaria chemoprophylaxis (impregnated mosquito net + efficient chemoprophylaxis in a malaria region), but recovery is very slow. In rare cases splenectomy is necessary. Steroids have no place in the treatment.

The disorder may be very similar to a certain indolent splenic lymphoma (e.g. splenic lymphoma with villous lymphocytes). The latter disorder is related to B-cell chronic lymphocytic leukaemia and occurs chiefly in elderly persons. The disease is often accompanied by significant cytogenetic abnormalities and monoclonal “villous” B-lymphocytes in the peripheral blood. It is likely that in HMS, excessive stimulation of the B-lymphocytes by malaria antigens increases the risk that oncogenic mutation may occur, followed by clonal growth of these cells. The extent to which this aetiopathogenetic mechanism is similar to the MALT lymphomas (mucosa-associated lymphoid tissue) which are sometimes seen in chronic infection with Helicobacter pylori, is unclear.

linical picture, Burkitt’s lymphoma

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This very malignant tumour originating from B-lymphocytes generally presents with swelling of the jaw and mouth ulcerations (African endemic form), but may be located primarily in the abdomen or the brain (cosmopolitan, non-endemic form). There are no blast cells in the peripheral blood, but in a closely related form there is a leukaemic phase (ALL-type [acute lymphoblastic lymphoma]). Histologically a monotonously uniform picture can be recognised, consisting of small cells with round to oval nuclei containing two or more prominent nucleoli. The basophilic cytoplasm may contain clear fat vacuoles (Oil Red O positive). There are many mitoses. The histological picture is sometimes described as a starry sky. The stars are macrophages. Metastasis occurs chiefly to the brain and bone marrow. The disease occurs almost exclusively in children. Infection with the Epstein-Barr virus (cf. mononucleosis) probably plays an important part in the endemic form of Burkitt’s lymphoma. Epstein-Barr viral DNA is found in more than 90% of African Burkitt’s lymphomas. Only one protein, EBNA-1, [Epstein-Barr nuclear antigen-1] is expressed. Repeated malaria attacks may have a mitogenic effect on infected B-lymphocytes increasing the risk of degeneration. The reciprocal chromosomal translocation 8q24  14q32 is considered as typical. A cellular proto-oncogene (c-myc) of chromosome 8 is hereby translocated to chromosome 14, next to the genes coding for the heavy chains of immunoglobulins. The proto-oncogene can also be translocated either next to the kappa genes (κ) coding for the light chains on chromosome 2 or the lambda genes (λ) for the light chains on chromosome 22. This repositioning to chromosome 2, 14 or 22 disturbs the control of the c-myc gene and gives rise to malignant growth behaviour. [Translocations of the gene bcl-2 from its normal place on chromosome 18 to a place next to the genes for the heavy chains on chromosome 14 is also sometimes found in the related follicular lymphoma. Normally the gene product bcl-2 prevents cellular apoptosis (programmed cell death]). Nevertheless the pathogenesis of Burkitt’s lymphoma is not yet completely clear.

The tumour can be treated with cytostatic drugs. Good results with IV cyclophosphamide (Endoxan®) among other drugs, are not unusual (the target dose 1-1.5 gram/m² IV every 3-4 weeks with 2 doses in remission). The alkylating cyclophosphamide should not be confused with the immunosuppressive cyclosporin! During treatment gout and haemorrhagic cystitis sometimes occur as complications. Allopurinol protects from the risk of hyperuricaemia during tumour lysis. If available, methotrexate (15 mg/m² P.O. for 3 days) and vincristine (1.5 mg/m² IV per week) may be given in addition. Citrovorum factor (leukovorin) is not necessary during brief administration of methotrexate. Combinations of several cytostatic drugs are sometimes used, e.g. CHOP or hyper-CVAD, cytarabine, L-asparaginase. CHOP consists of: cyclophosphamide 750 mg/m² IV day 1, doxorubicin 50 mg/m² IV day 1, vincristine 1.5 mg/m² mg IV day 1 and prednisolone 100 mg/m² per day for 5 days. Neurotoxicity such as polyneuritis during use of Vinca alkaloids is an inherent danger. Bone marrow suppression should be carefully monitored. Doxorubicin is cardiotoxic. Surgical debulking is sometimes carried out. Monoclonal antibodies (Mabthera®, Rituxan®) aimed at the B-cell specific CD-20 antigen which is expressed by 95% of all B-cell lymphomas, are almost never available but can be used in low-grade lymphomas. In the differential diagnosis, the possibility of a deep mycosis (e.g. rhinophycomycosis, mucormycosis) or a chronic abscess of the tooth, jaw or sinuses should be borne in mind.

Clinical picture, nephrotic syndrome in P. malariae

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Chronic infection with P. malariae may, via immunological mechanisms (chronic immune complex glomerulonephritis) cause a nephrotic syndrome, characterised by oedema and proteinuria (> 3.5 gram per 24 hours). There is often significant hyperlipidaemia, and lipid bodies are sometimes found in the urine (which appear in polarisation microscopy like bunches of grapes with a Maltese cross pattern). If a kidney biopsy is carried out, it should be borne in mind that severe bleeding will occur in 1% of cases. The treatment of nephrotic syndrome is difficult. A curative malaria treatment is of course indicated, but will not produce any improvement of the kidney problems. Salt restriction and diuretics are indicated (both thiazide and loop diuretics). Albumin IV and treatment with an ACE-inhibitor [angiotensin-converting enzyme-inhibitor] such as enalapril (Renitec®) is only possible in better settings. In significant hypercholesterolaemia simvastatin is beneficial but expensive (Zocor®, a HMG-CoA reductase inhibitor). Patients with advanced nephrotic syndrome lose coagulation inhibitors through the urine (protein S, C, antithrombin III) and are therefore at increased risk of thrombosis, chiefly in the vena renalis. Steroids and immunosuppressives are of little benefit in this disorder. An important challenge is to distinguish the entity from minimal change glomerulonephritis (electron microscopy needed to confirm "minimal change").

Malaria: Diagnosis, general

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When can one assert that someone has the disease "malaria"?

There are several problems and the question has still not been fully resolved. The demonstration of malaria parasites in the blood is essential, but insufficient in itself. Many people will develop an acquired immunity after several years of exposure, and may harbour parasites without exhibiting symptoms. The degree of parasitaemia may help, but there is no absolute criterion (the higher the parasitaemia, the more chance that malaria is in fact the diagnosis). There are patients with malaria for whom the thick smear is negative (luckily this is rare in a good laboratory). There are no pathognomonic clinical signs. An accurate diagnosis is becoming more and more important, in view of the increasing resistance of P. falciparum and the high price of alternatives to chloroquine.

Malaria: Diagnosis, clinical aspects

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No single clinical sign permits the diagnosis of malaria. Yet malaria must always be considered in cases of fever in the tropics. Since the symptoms can be quite diverse, a clinical diagnosis is unreliable in itself. Microscopic confirmation of the diagnosis is often not possible in many regions and situations. It is of the greatest importance that other important diagnoses are ruled out before instituting a blind anti-malaria therapy. All too often fever is considered as malaria without considering alternative diagnoses.

The presence of parasites does not rule out an additional diagnosis: e.g. someone with fever may well have some malaria parasites in a thick smear, but this does not rule out meningitis or pyelonephritis. Chronic carriers are people who, in spite of the fact that they have malaria parasites in their blood, have no symptoms of this. When such people develop another infection their symptoms are often attributed to the malaria parasites in their blood, although these are not responsible. The absence of parasites in a single preparation does not rule out malaria, but does make the diagnosis of P. falciparum highly improbable. Where there is strong clinical suspicion it is best to repeat the test 12h later.

Thick smear

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A thick smear concentrates the parasites 10 to 25 times. It is rather more difficult to interpret than a thin smear preparation and often does not permit species identification.

A thick smear contains no intact red blood cells (haemolysis due to the distilled water used in the staining). If a thick smear is positive, a thin smear should be examined. Sometimes parasitaemia is estimated in a thick smear and expressed as +, ++, +++. This form of record is of course quite subjective and confusing and is best avoided. If the thick smear is positive, it is then best to count the percentage of parasited cells in a thin smear preparation. The parasitic density can also be roughly determined in a thick smear, by counting the number of parasites per 200 leukocytes and multiplying this by 30. It is assumed that on average there are 6000 leukocytes per µl blood and that there is one leukocyte per 500 red blood cells. For example: 5 parasites per leukocyte (1000 parasites for every 200 leukocytes) corresponds to a density of 30,000 parasites per µl. Roughly 30,000 parasites per µl corresponds to a parasitaemia of 1% (a moderately anaemic person). If the thick smear is found to be negative in a reliable laboratory, and if there is nevertheless strong suspicion of malaria, the test is repeated every 12 hours for 48 hours. One great disadvantage of the thick smear method is that reliable technical expertise is needed which should be monitored (e.g. quality control). The argument that a lab technician has carried out the test for years and thus has plenty of experience, is absolutely no guarantee of quality or reliability. The test also requires plenty of time if the parasitaemia is low, or before a negative result can be concluded.

Thin blood smear

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This shows the presence of undistorted parasites. The thin blood smear permits identification and also calculation of the parasitaemia (% of parasitised red blood cells). This is necessary to start appropriate therapy (P. vivax is treated differently from P. falciparum). If the parasite cannot be identified it is regarded as a P. falciparum as a safety precaution. Mixed infections do occur. For staining, Giemsa is used with a slightly alkaline pH. It is a good habit to prepare the buffer solutions each day in the morning (interaction with CO₂ from the atmosphere changes the pH of older solutions). Phosphate buffers are mostly used.

First a stock solution is made of KH₂PO₄. This is made by placing 9.078 g KH₂PO₄in one litre of purified water. This stock solution can be used for weeks if correctly stored (in a closed bottle).
A stock solution of Na₂HPO₄.2H₂0 is also made by mixing 11.877 g with one litre of purified water. If one uses the anhydrate (Na₂HPO₄) in place of Na₂HPO₄.2H₂0, only 9.474 g per litre is used. This stock solution can also be used for weeks if correctly stored (in a closed bottle).
To obtain a buffer with a pH of 8, add 5.5 ml of KH₂PO₄ solution to 94.5 ml of Na₂HPO₄.2H₂0 solution and dilute with 900 ml of distilled water. One then has 1 litre of buffered water with a pH of 8. This can then be used for the malaria blood smears for the rest of the day.
For the leukocytic formula it is best to stain with a slightly acid pH of 6.4. For this a different phosphate buffer is used. This requires different ratios. Now 26 ml of the Na₂HPO₄.2H₂0 solution is mixed with 74 ml of the KH₂PO₄ solution and then 900 ml water is added.

An alternative to Giemsa is acridine orange, but the day-to-day use of this technique is quite unpleasant and it is necessary to have a special microscope (ocular filter, halogen light, interference filter above the condenser). [This latter filter restricts transmission to some narrow spectral bands].

P. falciparum infection is characterised by:

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small ring shapes, sometimes double chromatin specks
accolé forms (parasites adherent to the membrane of the red blood cell)
several parasites per red blood cell
few or no schizonts
banana-shaped gametocytes (the name "falciparum" comes from L. "falx" = sickle and "pario" = bring forth; they are sickle-shaped). Sometimes the red blood cell also contains inclusions (Garnham’s bodies) as well as the gametocyte.
High parasitaemia may occur in P. falciparum and is unusual in the other forms (parasitaemia > 2 % is suggestive of P. falciparum).

P. vivax preferably penetrates young (therefore large) erythrocytes.

P. ovale is often found in a thin smear preparation in rather oval-shaped, sometimes distorted red blood cells.

P. malariae trophozoites sometimes have a typical band shape. The mature schizonts have a daisy head appearance.

In well-stained preparations the nuclei of the parasites are always stained red and the cytoplasm blue. The presence of malaria pigment is very characteristic of the older stages of Plasmodium sp. P. falciparum often contains a single black dot. P. vivax often contains countless fine golden yellow/brown specks of malaria pigment. In P. ovale and P. malariae the pigment inclusions are many and brownish black. Countless fine red spots in the red blood cell (Schüffner’s dots) can be seen in P. vivax and P. ovale (the more mature the parasite, the more dots). In P. ovale the dots are sometimes called James’s dots. Sometimes a few flecks can be observed in P. falciparum (Maurer’s dots or clefts). P. malariae almost never exhibits dots (Ziemann’s dots). The visibility of these dots depends to a great extent on the acidity (pH) with which the thin slide preparation is stained (slightly alkaline: pH = 8 is best). The acidity is important because blood smears are usually stained for haematological tests with a slightly acid pH. With such a stain, the dots will not be seen clearly if at all.

Ratio gametocytes/trophozoites

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In an infected person, countless parasites can often be seen in the peripheral blood. Since gametocytes are the only forms which are responsible for transmission in nature, it is remarkable how few gametocytes are generally found. Often the number of gametocytes is only a small percentage of the number of trophozoites. Yet higher gametocyte densities must lead to higher transmission. Why gametocytes occur in such low proportions compared to the number of trophozoites is still not clear. Generally there are more female than male gametocytes, certainly early on in an infection. This is explained by the fact that one male gametocyte can form 8 viable male gametes, unlike the female gametocyte. This is in fact the case when infections are monoclonal, yet in mixed infections the ratio would have to be 1/1 according to population genetics. Later in the infection agglutinating antibodies are produced which immobilise male gametes and thus inhibit their function. Agglutination of female gametes has no effect on their function. To compensate for this the parasite produces more male gametocytes later in infection, possibly under the influence of increasing concentrations of erythropoietin. The latter hormone increases as anaemia increases. A single haploid clone of P. falciparum can produce both male and female gametocytes. Precisely how this works is not clear. Plasmodium falciparum gametocytes need 7-10 days for maturation. The sex ratio is determined by the erythropoietin content 7-10 days before they mature. It is interesting to note that P. falciparum exhibits increased infectibility in humans with sickle cell anaemia, regardless of the gametocyte density. This is explained by the chronically increased erythropoietin in this disorder, which leads to a higher percentage of male gametocytes. Nevertheless much study is still needed before the details will be fully understood.