{"id":1436,"date":"2020-03-20T22:29:12","date_gmt":"2020-03-20T22:29:12","guid":{"rendered":"https:\/\/haema-journal.gr\/?p=1436"},"modified":"2020-03-23T22:44:03","modified_gmt":"2020-03-23T22:44:03","slug":"microscopical-diagnosis-of-blood-parasites","status":"publish","type":"post","link":"https:\/\/haema-journal.gr\/?p=1436","title":{"rendered":"Microscopical Diagnosis of Blood Parasites"},"content":{"rendered":"

Haema 2019; 10(2):68-112
\n<\/em><\/p>\n

Konstantinos Liapis<\/p>\n

Consultant Haematologist, Georgios Gennimatas Hospital<\/p>\n

Full PDF<\/a> | \"\"<\/a><\/p>\n

<\/p>\n

Blood is examined for the following parasites:<\/p>\n

– Plasmodium
\n– microfilariae
\n<\/em>– Trypanosoma
\n-Leishmania<\/p>\n

\n

\u039dote: in some areas, malaria, microfilariae and\/or trypanosomes may all be present and, therefore, it is not unlikely to detect two parasites e.g. Loa loa and P. falciparum in the blood film of a patient from Nigeria or Cameroon.<\/p>\n

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Blood films are critical in the diagnosis of malaria, filariasis, and trypanosomiasis. Bone marrow aspiration is important in the diagnosis of leishmaniasis and occasionally leads to the diagnosis of other parasitic infections. In rare cases, leishmania organisms may be seen in the granulocytes or monocytes of the peripheral blood.<\/p>\n

\u03a4hin films or thick films?<\/em><\/strong><\/p>\n

Either thin films or thick films may be used depending on the circumstances. \u03a4he thick film is more sensitive in detecting plasmodia and trypanosomes. Examine the centre portion of the thick film first (where it is more likely to detect parasites), and then examine the thinner outer edges (where the morphology is better). In thick films, organisms tend to be more compact and denser than in thin films (they often appear smaller). In contrast, the thin film causes very little distortion of the parasite, and permits species identification when it may not be possible in thick films.<\/p>\n

The morphological features of malaria parasites may not be distinct in the thick film and, therefore, the thin film should be used for species identification.<\/p><\/blockquote>\n

\n

Note:<\/p>\n

– in most cases of malaria, the thin film will be positive. In order to increase sensitivity, look carefully in the thicker areas of the thin film (a \u2018form\u2019 of thick film).
\n– 3 consecutive negative thin films practically rule out malaria (repeat every 12-24 hours up to three; attempt to obtain blood samples during fever)
\n– if P. falciparum is seen, you should request a thick film for calculation of parasitaemia (parasites per \u03bcl \u03bff blood).
\n– thick films should be used when searching for microfilariae (unlike Plasmodium and Trypanosoma, thick films cause no distortion of the morphological chracteristics of microfilariae).<\/p>\n

\n
1.1 MALARIA PARASITES1-12<\/sup><\/strong><\/h5>\n

See \u2013 Cases illustrating malaria parasites in: Illustrations of Blood Parasites (Figures 28-38)<\/p>\n

See \u2013 Life cycle, epidemiology, and clinical information in Supplementary Appendix 1<\/p>\n

Diagnosis of malaria is based primarily on the recognition of parasites in well stained thick and thin blood films stained with a Romanowsky stain (e.g. Giemsa, Leishman, Field) at pH 7.2-7.4. Thick films may improve sensitivity (it is said that the limit of detection of plasmodia is 10 parasites per mL blood versus 200 parasites per mL blood for thin films) but, as mentioned, a thin film is preferable for species identification.<\/p>\n

In examining thin blood films for malaria, you must look at the appearance of the red cells containing the parasites and the appearance of the parasite.<\/p>\n

1. Infected red cells<\/strong><\/p>\n

– Look at the size of the infected red cells
\n– Is the red blood cell filled with pink or red staining dots (Sch\u00fcffner\u2019s dots)?<\/p>\n

\"\"<\/p>\n

* Not always! P. vivax or \u03a1. ovale parasitised red cells may be normal sized, especially when they contain young trophozoites (rings).
\n** Sch\u00fcffner\u2019s dots are called James\u2019 dots in the case of \u03a1. ovale.
\n*** Sch\u00fcffner\u2019s dots may not be present in the early rings of P. vivax or \u03a1. ovale. Also, in poorly stained slides, Sch\u00fcffner\u2019s dots may not be visible so it is essential that correct staining methods are used. In particular, the pH of the buffered saline used in the Giemsa stain has a profound effect on the staining of Sch\u00fcffner\u2019s dots and parasites.<\/p>\n

Sch\u00fcffner’s stippling versus basophilic stippling \u2014 what is the difference? 1. Sch\u00fcffner\u2019s dots are pink or red staining dots; basophilic stippling is blue or blue-purple 2. Unlike basophilic stippling, Sch\u00fcffner\u2019s dots will not be present in red cell without parasites.<\/p>\n

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Note: the appearance of Sch\u00fcffner\u2019s dots depends on the quality of the Giemsa stain.<\/p>\n

\n

The pH of the buffered saline used in the Giemsa stain is usually 6.8. The desired standard for malaria staining is 7.2. Sch\u00fcffner\u2019s dots may not be visible in slides stained with Giemsa at pH <6.8 (Figure 1).<\/p>\n

Red cell stippling does not occur in P. malariae. However, fine pink-staining dots (called Ziemann’s dots) may appear in alkaline pH \u22657.5 or after prolonged staining. Ziemann\u2019s dots do not appear if correct staining methods are used.<\/p>\n

Tip:<\/strong> a simple, practical way to evaluate pH are the eosinophilic granules which are excellent pH markers because they stain bright red when the reagents are acidic and blue or dark grey when they are alkaline. Also, erythrocytes stain orange in acidic pH <6.4 and green in alkaline pH >7.6. In properly stained slides, eosinophilic granules stain red-orange and erythrocytes reddish or red.<\/strong><\/em><\/p>\n

Reliable and accurate parasite identification depends on good quality Giemsa.<\/p>\n

Poor quality Giemsa may lead to technical errors.<\/p>\n

Be sure to use newly prepared Giemsa. Don\u2019t use Giemsa of dubious pedigree!<\/p><\/blockquote>\n

Parasites<\/strong><\/p>\n

Identification of malaria parasites<\/strong><\/em><\/p>\n

Things that may be confused with malaria parasites in blood films include platelets adhering to red blood cells, superimposed platelets Cabot\u2019s rings, Howell-Jolly bodies, precipitated stain on red blood cells, unfiltered or contaminated Giemsa, debris from the patient\u2019s skin, dust that gets on the blood film while it is drying and other artefacts that may appear on the slide during preparation. All these may look like malaria parasites in stained blood films and can be mistakenly identified as malaria. Usually, however, they do not have all three components of a malaria organism (i.e. the basic structure of malaria parasites).<\/p>\n

The three components of malaria parasites are:<\/p>\n

– blue staining cytoplasm
\n– red or purple staining chromatin
\n– brown or black pigment (haemozoin).<\/p>\n

Except for early (young) rings (which lack pigment), you should see all three characteristics before you identify any structure as a malaria parasite. If you are uncertain whether a particular object or structure is a malaria parasite, look for more typical forms.<\/p>\n

Table 1<\/a> shows the characteristics of the four main plasmodium species.<\/p>\n

\"\"<\/p>\n

Figure 1.<\/strong> Effect of pH on Giemsa staining of malaria parasites.<\/p>\n

Figures 2-5 are modified from: \u0392ench aids for the diagnosis of malaria, WHO, 1988. Also, see selected cases illustrating malaria parasites in: Illustrations of Blood Parasites (Figures 28-38).<\/p>\n

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Figure 2.<\/strong> Plasmodium ovale (parasitaemia is often not high).<\/p>\n

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Figure 3.<\/strong> Plasmodium vivax.<\/p>\n

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Figure 4.<\/strong> Plasmodium falciparum.<\/p>\n

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Figure 5.<\/strong> Plasmodium malariae (it is associated with low-level parasitaemia \u22641%).<\/p>\n

Maurer\u2019s dots versus Sch\u00fcffner\u2019s dots \u2014 what is the difference? Maurer\u2019s dots are bigger, darker, and far less numerous than Sch\u00fcffner\u2019s dots (the red cell is not entirely filled in contrast to Sch\u00fcffner\u2019s stippling).<\/p>\n

Rings of the four species may look alike. If you see rings, look for older stages (late trophozoites, schizonts, and gametocytes) \u2192 older stages should help you identify the Plasmodium species.<\/p>\n

In patients with P. falciparum only rings are usually seen (or rings + gametocytes or only gametocytes); older stages (schizonts) are present only in severe infections. Occasionally, late trophozoites may be seen.<\/p>\n

P. falciparum schizonts are rare in peripheral blood films (only in severe infections with high parasitaemia) because schizonts sequester in the microcirculation (through small protuberances or knobs of the erythrocytic membrane) causing obstruction. Pfemp-1, an important cell adhesion molecule on knobs, interacts with ICAM-1 on endothelial cells (\u2192 sequestration) and with CD36 on adjacent red cells (\u2192 r\u03bfsetting).<\/p>\n

In contrast, late trophozoites and mature schizonts are common in blood films of patients with other malaria species.<\/p>\n

How to identify trophozoites, schizonts, and gametocytes:<\/p>\n

Schizont \u2192 a schizont contains \u22652 nuclei (because schizogony has occured)<\/p>\n

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Note: the nucleus of P. falciparum or P. vivax rings may include two chromatin dots (like \u2018headphones\u2019).<\/p>\n

\n

Trophozoite \u2192 contains 1 nucleus and vacuole (usually)<\/p>\n

Gametocyte \u2192 contains 1 nucleus without vacuole<\/p>\n

P. falciparum \u2192 (usually) rings and\/or gametocytes present in peripheral blood (rarely: late trophozoites, schizonts)
\nP. falciparum gametocytes appear in the peripheral blood after approximately one week of fever.
\nP. vivax \u2192 (usually) all stages present in the peripheral blood.<\/p><\/blockquote>\n

If<\/strong> P. falciparum<\/em><\/strong> is seen, the level of parasitaemia must be reported. <\/strong><\/p>\n

You can estimate the level of parasitaemia by three methods:<\/p>\n

    \n
  1. % parasitaemia (number of infected red cells per hundred)<\/li>\n<\/ol>\n

    Using 100\u00d7 (oil immersion) objective, count the number of infected erythrocytes in 5 fields of a thin film (~1000 erythrocytes).<\/p>\n

      \n
    1. parasitaemia\/\u03bcl (parasites per microlitre)<\/li>\n<\/ol>\n

      The following is a practical method of adequate accuracy. This method is based on the number of parasites per \u03bcl of blood in a thick film, these being counted in relation to a predetermined number of leukocytes. Two tally counters are required to count parasites and leukocytes separately in a thick film. Using 100\u00d7 (oil immersion), count the number of parasites (P) and leukocytes until 300 leukocytes have been enumerated.<\/p>\n

      Parasitaemia (\/\u03bcl) = W\u0392C \u00d7 P\/300
      \n(W\u0392C = white blood cell count)<\/p>\n

      Count all the species present (some cases are mixed infections) and include both sexual (gametocytes) and asexual forms together.<\/p>\n

      This method must always be used in cases of suspected drug-resistant parasites.<\/p>\n

        \n
      1. The plus system using the 100\u00d7 (oil immersion) objective in a thick film:<\/li>\n<\/ol>\n

        +\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 = 1-10 parasites\/100 fields<\/p>\n

        ++\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 = 11-100 parasites\/100 fields<\/p>\n

        +++\u00a0\u00a0\u00a0\u00a0\u00a0 = 1-10 parasites\/1 field<\/p>\n

        ++++\u00a0\u00a0 = >10 parasites\/1 field<\/p>\n

        The plus system is not very accurate and should be used only when it is not possible to undertake the more acceptable parasite count per \u03bcl of blood.<\/p>\n

        The parasitaemia\/\u03bcl method and the plus system are based on the estimation that about 100 fields, using a 100\u00d7 (oil immersion) objective, is the equivalent of 0.25 \u03bcl of blood.<\/p>\n

        Haematological changes associated with malaria:<\/strong><\/p>\n

        Malaria may be accompanied by atypical (reactive)<\/strong> lymphocytes<\/strong> in the blood film (usually <15% of the leukocyte count \u2260 infectious mononucleosis). It is not uncommon to find plasmacytoid lymphocytes (plymphs) and T\u00fcrk cells and you may even find circulating immunoblasts (activated large \u0392-cells) with deeply staining basophilic cytoplasm, high nuclear:cytoplasmic ratio, and nucleoli (Figure 6).<\/p>\n

        \n

        Note:<\/p>\n

        lymphoplasmacytoid cells may be seen in the peripheral blood in cases of immune system activation by various stimuli.<\/p>\n

        reactive lymphocytosis is more common and prominent in another tropical disease that is very similar to malaria, dengue, which is endemic in South-East Asia, western Pacific, and the Caribbean.<\/p>\n

        \n

        \"\"<\/p>\n

        The neutrophil count<\/strong> is usually low or low-normal (due to margination to the endothelial surface), normal, or slightly increased. The platelet count<\/strong> is often reduced in acute malaria (the mean platelet count is 132\u00d7109<\/sup>\/l at presentation and rises to 234\u00d7109<\/sup>\/l, 12 days after treatment), however, severe thrombocytopenia (<40\u00d7109<\/sup>\/l) is rare (5%). Several mechanisms are responsible for the thrombocytopenia: splenic pooling, platelet activation, platelet binding to CD36 on infected red cells, and antibody mediated platelet destruction. Megakaryocytes in the marrow are usually normal or increased in number (i.e. peripheral thrombocytopenia).<\/p>\n

        Note: platelets are often abnormally large in malaria (e.g. MPV 10.5 fl) so that the automated platelet count may be falsely low.<\/em><\/strong><\/p>\n

        A major mechanism of haemolysis<\/strong> in malaria is the rupture of red cells at the time of release of merozoites (i.e. haemolysis in malaria is linked to the red cell infection and is related to the level of parasitaemia). Coombs\u2019 test is usually (-). However, P. vivax<\/em> may rarely cause Coombs (+) warm autoimmune haemolytic anaemia with spherocytosis, and some antimalarial drugs can cause haemolytic anaemia in individuals with G6PD deficiency. Artemisin, the drug of choice for severe malaria, may rarely cause severe intravascular Coombs (-) delayed haemolytic anaemia, occurring 7-31 days after treatment. The mechanism is unknown.<\/p>\n

        During acute malaria there is immobilisation of iron<\/strong> in the macrophages, and low serum iron concentrations: serum ferritin is increased (acute reactive protein). Serum folic acid<\/strong> is normal or low. Red cell folate levels are raised above normal.<\/p>\n