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Do you know which disease fits this month’s case? Then test your knowledge in the quiz below!

Can you explain the mild thrombocytopenia in this patient? Idiopathic thrombocytopenic purpura (ITP)
Malaria caused by Plasmodium vivax infection
Rheumatoid arthritis
Pneumonia

Online Version of this month´s case:

Correction

It has been brought to our attention that the schizont, visible on the peripheral blood smear, is too large and contains too many merozoites for a Plasmodium malariae infection. Follow up confirmed that this was a Plasmodium vivax infection. We apologize for the confusion.

The correct answer to January´s Quiz is:

Malaria caused by Plasmodium vivax infection

 

 

Scattergrams and microscopy

Table

Interpretation and Differential Diagnosis

The answer can be inferred from…

  • Presence of the action message ‘Difference between WNR and WDF. Check the results’, caused by differences in WBC counts from the WDF channel (9.53 x 109/L) and WNR channel (5.64 x 109/L)
  • Presence of an abnormal neutrophil population in the WDF scattergrams: increased neutrophil granularity (NEUT-GI) and two neutrophil populations in the SSC-FSC scattergram
  • Unreliable results for all WBC differential parameters, caused by the presence of the abnormal neutrophil population
  • Combination of relative neutrophilia and normal neutrophil reactivity (NEUT-RI)

Case history

A 34-year old man who frequently travels to Papua New Guinea visited his physician with a fever, nausea, headache and painful joints. Considering the man’s travel history, a complete blood count with WBC differential and reticulocyte analysis was performed to investigate a possible malaria infection.

Case results

Absolute neutrophil counts were normal but close to 95% of all WBC in the peripheral blood were classified as neutrophils indicating a relative neutrophilia. Furthermore, the Neutrophil-Granularity-Intensity (NEUT-GI) was increased while the Neutrophil-Reactivity-Intensity (NEUT-RI) was normal. Interestingly, the SSC-FSC scattergram of the WDF channel showed two neutrophil populations and the WBC count from the WDF channel was much higher than the count from the WNR channel (9.53 x 109/L compared to 5.64 x 109/L). This is indicative of nucleic acid-containing cellular inclusions in RBC, which don’t interfere with the WBC count in the WNR channel due to the strong lysis reagent. Since RBC are not completely lysed in the WDF channel, they are visible as an additional neutrophil population. The presence of this abnormal neutrophil population in the scattergram without signs of neutrophil activation (normal NEUT-RI) pointed to a P. malariae infection. The negative Delta-He and low RET-He are inflammatory signs, while a mild thrombocytopenia is common in malaria (1) but also in the alternative diagnoses. Malaria was confirmed in the peripheral blood smear, which showed the presence of parasites in the early Schizont stage.

(*Please note that Sysmex offers for XN-Class analysers a new software that triggers a flag ‘iRBC?’ (inclusions in RBC) flag in such situations. An automated correction of the WBC count and the differential is part of the algorithm.)

The following answers are incorrect for the described reasons

 

Idiopathic thrombocytopenic purpura (ITP)

ITP is an autoimmune haematological disorder in which autoantibodies against platelet antigens induce accelerated platelet destruction, leading to a reduction in peripheral blood platelets. ITP causes a characteristic purpuric rash and a tendency to bleed, for example from the nose or periodontal gums. It is difficult to distinguish ITP from other causes of thrombocytopenia so its diagnosis is a process of exclusion. Megakaryopoietic activity of the bone marrow is typically enhanced in ITP patients but it was probably normal or decreased in the presented patient (normal MPV and P-LCR values) suggesting a production problem rather than accelerated platelet destruction. In addition, ITP patients don’t have a (pseudo)neutrophilia or ineffective erythropoiesis so an ITP was highly unlikely.

Rheumatoid arthritis

A rheumatoid arthritis (RA) diagnosis was also explored because the patient had painful joints. Like ITP, RA is also an autoimmune disease; RA-associated autoantibodies affect the lining of the joint cavities (synovium) in synovial joints. Over time this causes deformation of the joints leading to serious disability when left untreated. RA affects 1.1% of women and 0.4% of men. RA is associated with an inflammatory response, which was not seen here: WBC counts were normal and lymphocyte counts were decreased here.

Acute Legionella pneumonia infection

Acute bacterial infections, like a Legionella pneumonia infection, are characterised by increased granulation and activation of neutrophil granulocytes. NEUT-GI was increased here, which could point to hypergranulation associated with an infection. However, NEUT-RI is also increased in patients with bacterial infections because DNA transcription is increased during neutrophil activation, resulting in elevated cellular nucleic acids in the form of messenger RNA (mRNA). Consequently, staining of activated neutrophils with Fluorocell-WDF leads to an increased side fluorescence and high NEUT-RI. The WDF scattergram of this patient revealed a relative neutrophilia and an increased NEUT-GI but NEUT-RI was normal and therefore a Legionella pneumonia infection was unlikely.

Underlying Disease

Malaria

Despite different treatment options malaria remains a devastating infectious disease in the tropics: Each year 100-300 million people are infected with malaria worldwide and this is fatal in approximately 627,000 patients, mainly children below the age of five in Africa (2). While malaria exists in more than 100 countries it is mainly confined to poorer tropical areas of Africa, Asia and Latin America. More than 90% of malaria cases and the great majority of malaria deaths occur in tropical Africa. Older Africans have a reduced infection risk because they develop a degree of immunity as a result of continuous exposure. Pregnant women in Africa (especially women who are pregnant for the first time) are particularly vulnerable. Malaria poses a risk to travellers as well, and emigration from endemic areas also results in an increasing incidence in non-endemic areas (3). This poses a challenge because knowledge about diagnostics and treatment of malaria is not commonplace in Europe.

Malaria is a haemoprotozoan parasitic infection transmitted primarily by the bite of an infected female Anopheles mosquito. Malaria may also be transmitted via blood transfusion or congenitally between mother and foetus but this is rare. In humans, parasites multiply exponentially in the liver and, after several developmental stages, in infected RBC. Mosquitoes ingest parasites with a blood meal upon which the parasites undergo another reproductive phase inside the mosquito before being passed on to another human host.

Malaria species

Of the four species of Plasmodium that commonly infect humans (P. falciparum, P. vivax, P. ovale and P. malariae) P. falciparum accounts for most morbidity and mortality. It is found throughout tropical Africa, Asia and Latin America. Infection with P. falciparum is not restricted to RBC of a particular age and therefore results in the highest levels of parasitaemia. P. vivax is found worldwide in tropical and some temperate zones while P. ovale occurs mainly in tropical West Africa. These infections are similar to P. vivax infections but they are usually less severe and often heal without treatment. P. malariae also occurs worldwide but has a limited, intermittent distribution. Those infected with P. malariae remain asymptomatic for a much longer period of time than those infected with P. vivax or P. ovale and recrudescence is common.

Clinical manifestations

Fever and chills are almost always present (96% of patients) and are accompanied by headache (79%), muscle pain (60%), hepatomegaly (33%), splenomegaly (28%), nausea and vomiting (23%), and abdominal cramps/diarrhoea (6%). Clinical symptoms of malaria usually occur around the time of RBC lysis with fever resulting from the release of merozoites, malarial pigment and protein, and cellular debris. Chills or rigors followed by high fever occur in a cyclical pattern in P. vivax, P. ovale and P. malariae infections but not in P. falciparum infections, which exhibit a continuous fever with intermittent spikes. The classical malaria paroxysm is often not observed but it consists of three phases: the ‘cold’ stage, the ‘hot’ stage and the ‘sweating’ stage. Non-immune patients experience some or all of these symptoms, while persons living in endemic areas are infected intermittently and develop partial immunity, leading to less severe symptoms.

P. falciparum infection: P. falciparum parasites mature in RBC causing surface ‘knobs’ to form, which renders the RBC inflexible and ‘sticky’. These inflexible, ‘sticky’ RBC adhere to endothelial cells of capillaries and postcapillary venules in the brain, kidneys and other organs, obstructing blood flow. In the obstructed microvasculature parasites consume glucose, cause acidaemia and release tissue necrosis factor. The capillaries subsequently become more permeable allowing leakage of protein and fluids, which results in tissue oedema, anoxia, organ damage and death. In some cases infected RBC cannot be found in peripheral blood smears because they are sequestered in the host microvasculature. Severe complications of P. falciparum infection include cerebral malaria, renal failure, pulmonary oedema, gastro-enteritis (particularly in children) and anaemia.

P. vivax and P. ovale infection: P. vivax and P. ovale infections are similar. They are less severe than Falciparum-malaria and blood parasite levels are lower because only young erythrocytes are infected. Knobs do not develop on infected RBC and microvascular obstruction does not occur; therefore there is no damage to the brain, kidney, lung or other organs. Both parasites form a dormant stage in liver cells called hypnozoites, which can become activated and cause a delayed infection of RBC or relapse. Relapse usually occurs within 6 months of an acute attack but the period of dormancy may be much longer. Since fewer RBC are haemolysed their loss stimulates a reticulocyte response, which increases the number of young cells susceptible to infection. Fever is almost always present and may be erratic or continuous in the initial phase of the illness. When left untreated fever develops after three to four days, exhibiting a cycle of afternoon temperature increases every 48 hours, often as high as 40°C, and symptoms during this phase may be worse than in Falciparum-malaria. Physical findings may include an enlarged, tender spleen and a palpable liver. Occasional deaths have resulted from rupture of an enlarged spleen.

P. malariae infection: This is the mildest but most chronic of all forms of human malaria. RBC parasitisation occurs slowly so parasite levels are low and clinical features are usually mild. As in P. vivax and P. ovale infections, febrile outbursts develop in the afternoon but cycle every 72 hours. Patients may have several febrile cycles before parasites appear in the peripheral blood. P. malariae (and P. falciparum) do not have a dormant hypnozoite stage so relapses do not occur. Recrudescence can, however, occur many years after the initial infection. Low-grade infections can persist up to 20-30 years and splenomegaly is a common complication in such patients. P. malariae infection may produce a unique immune complex glomerulonephritis three to six months after transmission, which may lead to a renal failure.

Malaria in pregnancy: Because of the associated immune suppression, recrudescence and relapse are frequent during the second and third trimester. Malaria can also potentiate the anaemia of pregnancy, and cause acute renal insufficiency and hypoglycaemia (P. falciparum infections). It is associated with increased numbers of abortions, miscarriages, stillbirths and neonatal deaths.

Malaria in children: In non-immune children, common symptoms include drowsiness, anorexia, thirst, headache, nausea, vomiting and diarrhoea. Common early symptoms include increased temperature (>40°C), pallor and cyanosis; hepatomegaly and/or splenomegaly follow later. Cerebral malaria is the most frequent complication in children and convulsions are frequent. Anaemia is a complication with repeated infections. Children living in endemic areas develop limited immunity, which results in milder clinical symptoms that are more difficult to detect, including restlessness, loss of appetite, tiredness, sweating, intermittent fever and low-grade anaemia.

Laboratory findings

Normocytic, normochromic anaemia with leucocytopenia and thrombocytopenia may be present on initial screening. High-grade P. falciparum infection causes severe anaemia and an increase in the reticulocyte count. Traces of protein, urobilinogen and conjugated bilirubin may be found on urinalysis. In severe P. falciparum infections, massive haemolysis plus circulating immune complexes produce acute renal insufficiency or failure (‘blackwater fever’) with haemoglobinuria, proteinuria, elevated blood urea nitrogen (BUN) and elevated serum creatinine. If serum creatinine rises disproportionately higher than BUN (ratio >10-12:1), renal failure must be considered. While hyperbilirubinaemia usually results from haemolysis, impairment of liver function may also be indicated by increased liver enzymes (ALT, AST), prolonged prothrombin time and decreased serum albumin. This may cause diagnostic confusion with viral hepatitis. The hypoglycaemia, often present in P. falciparum infections results from increased consumption of glucose by parasitised RBC.

Malaria control and treatment

Between 2000 and 2012, mortality from malaria infections decreased by 42% while the number of infections dropped by 25% worldwide (2). Control has traditionally relied on:

  1. Control of the Anopheles mosquito vector by removal of breeding sites, use of insecticides and the use of insecticide-impregnated screens and bed nets;
  2. Diagnostic testing;
  3. Treatment with artemisinin-based combination therapy (ACT).

Although several candidate malaria vaccines are currently in clinical trials, a long hoped-for effective vaccine has not yet materialised and resistance to existing therapies and insecticides remains a concern. Treatment largely relies on antimalarial drugs, such as ACT for P. falciparum or chloroquine for P. vivax in areas where this drug is still effective (P. falciparum is resistant to chloroquine in most malaria-affected areas). In addition, sulfadoxine-pyrimethamine is recommended as a preventative therapy in pregnant women and children in endemic countries where parasite resistance to this drug is of little consequence. Artemisinin (4), extracted from sweet wormwood (Artemisia annua), has long been the most potent antimalarial drug available and it has been widely used to treat multidrug-resistant malaria. However, parasite resistance to artemisinin has been detected in Cambodia, Myanmar, Thailand and Vietnam so the World Health Organization (WHO) recommends gradually replacing artemisinin mono-therapy with ACT (2).

Literature

  1. Lampah DA et al. (2014): Severe Malarial Thrombocytopenia: A Risk Factor for Mortality in Papua, Indonesia. J Infect Dis: early online.
  2. World Health Organization (2013): World Malaria Report. www.who.int/malaria/publications/world_malaria_report_2013/report/en/
  3. Muentener P et al (1999): Imported malaria (1985–95): trends and perspectives. Bulletin of the World Health Organization 77: 560–565.
  4. Eckstein-Ludwig U et al (2003): Artemisinins target the SERCA of Plasmodium falciparum. Nature 424(6951): 957-961.

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