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Participants on the one-day teaching course are provided with a subject specific illustrated bench book. The following few pages are 'tasty tit-bits' extracted from the complete bench book. [Diagnosis] [Alternatives] [Field's] [Giemsa] [Changes] [Biochemistry] [Why?] [Trypanosoma] [Leishmania] Diagnosis
of malaria parasites Malaria continues to be
a great burden globally with >300 million acute cases contracted per annum,
accounting for >1 million deaths. The majority of these deaths occur in young
children under the age of 5 years (one child dies every 40 seconds) in
sub-Saharan Africa. In 2006, 1,758 cases of malaria were reported to the Malaria Reference Laboratory. Of these 1,386 were Plasmodium falciparum . The largest proportion of travellers returning to the UK
with malaria are those who have been visiting friends or relatives. Suspected
malaria is a medical emergency.
Sampling and processing of the blood sample must not be delayed if malaria is suspected. Malaria parasites may be present in the peripheral blood even in
the absence of fever. Therefore, if a clinical suspicion of malaria exists,
collection of a blood sample is mandatory, whatever the patient’s temperature. When it is necessary to use
anticoagulants, films should be made as soon as possible, certainly less than 2
hours after the blood was drawn. EDTA is superior to other anticoagulants for
this purpose. Parasite and red cell morphology can be seriously affected if the
blood has been in anticoagulant for too long. Development of the sexual
stages of the parasite may occur (even within 20 minutes) and the male gametes
released into the plasma may be mistaken for other organisms such as Borrelia
or Leptospira.
Staining Giemsa
(BDH R66 formulation) staining of malaria parasites in thin blood smears at pH
7.2 provides the best conditions for the demonstration of parasite morphology
and the presence or absence of red cell inclusions. Giemsa
is sold as a concentrated stock solution. Each new batch number should be tested
for optimal staining of red cells and length of staining time prior to use. The
ideal control is a fresh blood film containing Plasmodium vivax. In the absence of fresh material, methanol fixed
films stored with silica gel in a sealed, airtight container at 40C
may be substituted. Failure to demonstrate Schòffner’s
dots might lead to misdiagnosis, and a stain, which fails to achieve this on a
known Plasmodium vivax, must be discarded. Alternative
detection methods. In 2000 the WHO produced a document entitled New Perspectives in Malaria Diagnosis (WHO/MAL/2000.1091) Certain recommendations were presented on what non-microscopic rapid diagnostic tests (RDT) should provide. The document concluded that 'results from these test devices should be at least as accurate as results derived from microscopy performed by an average technician under routine field conditions'. Other criteria included the sensitivity, which should be above 95% compared to microscopy, and the detection of parasitaemia, such that levels of 100 parasites/µl (0.002% parasitaemia) should be detected reliably with a sensitivity of 100%. HRP2 detection. A monoclonal antibody of high affinity against P. falciparum histidine-rich protein 2 (HRP2), found in the membrane of infected erythrocytes and in the plasma of infected persons has been used in commercially available tests such as ParaSightÔ, ICT Pf or Pf/Pv and PATH Falciparum Malaria IC test. HRP2 - based immunochromatographic tests permit rapid diagnosis of P. falciparum malaria, their clinical usefulness for the diagnosis of other Plasmodium spp. and for monitoring of the therapeutic response is limited. Since HRP2 is expressed only by P. falciparum, these tests will give negative results with samples containing only P. vivax, P. ovale or P. malariae. pLDH
detection. pLDH is a soluble glycolytic enzyme expressed at high levels in asexual stages of malaria. Sensitivity for RDT remains a problem and a negative RDT cannot at present be accepted at face value. Many laboratories have found that these RDT provide a very useful backup for inexperienced microscopist during night calls. However, it must be emphasised that the microscopic examination of correctly stained thick and thin blood films by adequately trained staff is still considered to be the WHO gold standard. [Index] Field’s stain is made
from two component stains. Field’s stain A and Field’s stain B. By using
buffers of differing pH it is possible to stain a wide variety of clinical
materials Field’s
stain for thick blood films. 1. Air dry the thick
film until it is completely dry, failure to do so will result in the blood 2. Dip the slide
vertically into undiluted Field’s stain A for 3 seconds. 3. Gently rinse the
slide in tap water for 3 seconds. 4. Dip the slide into
undiluted Field’s stain B for 3 seconds. 5. Wash gently in tap
water for a few seconds until the excess stain has run out. 6. Drain vertically to
dry. 7. Examine with the X100
oil immersion lens. When searching for malarial
parasites the slide/slides should be examined for at least 15 minutes or 200
fields before declaring the slide/slides negative. For further reading
participants are advised to acquire a copy of the following publications; ACP
Broadsheet No. 148 July 1996 Laboratory diagnosis of malaria
D
C Warhurst and J E Williams The
laboratory diagnosis of malaria. Prepared by the Malaria
Working Party of The General Haematology Task Force of the British Committee for
Standards in Haematology Clin. Lab. Haem. 1997. 19
165-170
Notes on reading
thick films for malarial parasites. ·
The end of the film at the top of
the slide when it was draining should be looked at. The ideal area is to be
found where the slide appears to be a pink/blue colour. In this area, the
background should appear as a pale grey/blue colour and the nuclei of the white
cells are purple in colour. This gives better differentiation of the stained
parasites. The edges of the film will also be better than the centre, where the
film may be too thick and cracked. 200 fields must be examined before the film
is declared negative. ·
Thick films are unfixed,
therefore the red cells lyse when dipped into water, there can be no accurate
identification of the parasites present based on the size, shape and stippling
of the red cells. Occasionally a ghosting effect may be seen due to the fine
stippling of Schüffner’s/James’s dots. ·
Schizonts and gametocytes if
present should be easily recognisable. ·
White cells, platelets and
malarial pigment can also be seen on the thick film. · In a well-stained film, the malarial parasites show deep red chromatin and pale blue cytoplasm. The white cell nuclei are purple and the background is a pale grey/blue colour.
Rapid
Field’s stain. This is a modification of
the original Field’s stain to enable rapid
staining of fixed thin films. This
method is suitable for malaria parasites, Babesia sp., Borrelia sp. and
Leishmania sp. Method. 1. Air dry the film 2. Fix in methanol for 1
minute. 3. Flood the slide with
1 ml of Field’s stain B, diluted 1 in 4 with 4.
Immediately, add an equal
volume of undiluted Field’s stain
A, mix well and allow to stain for 1 minute. 5. Rinse well in tap
water and drain dry. Uses. This is a useful method for
rapid presumptive species identification of malarial parasites. It shows
adequate staining of all stages including stippling (mainly Maurer’s clefts).
However, staining with Giemsa is always the method of choice for definitive
species differentiation. Method. 1. Air dry thin films 2. Fix in methanol for 1
minute 3. Wash in tap water and
flood the slide with Giemsa diluted 1 in
10 with 4. Stain for 25 - 30
minutes. 5. Run tap water on to
the slide to float off the stain and to prevent deposition of precipitate on to the film. Drain dry vertically. 6. Examine the
film using the x100 objective. Notes
on the stained film.
On a well-stained film the
chromatin stains red/purple and the cytoplasm blue. Leucocytes have purple nuclei, the red stippling, if present should be clearly visible. [Index] Malaria
- haematological changes. 1.
Anaemia. ·
Due to parasite schizogony ·
Immune haemolysis – due to the
formation of IgG antibody against the parasites with non-specific attachment of
the immune complexes to the red cell membrane with complement activation and
subsequent phagocytosis. ·
IgG coated red cells cleared more
rapidly by spleen ·
Maturation defects in marrow 2.
Thrombocytopaenia. ·
Platelet survival time reduced to
2-4 days ·
Increased number of large abnormal
megakaryocytes found in marrow ·
Circulating platelets may be
enlarged suggesting dyspoietic thrombopoiesis ·
Enhanced splenic up take or
sequestration ·
Patients with DIC, platelets may
be removed from circulation at sites of thrombin deposition 3.
Mild leucopaenia. ·
Frequently described in
uncomplicated malarias. Cause unknown. 4.
Neutrophil leucocytosis. ·
Important abnormality in patients
with severe P. falciparum malaria. TNF may be responsible for the leucocytosis
that may be associated with a complicating bacteraemia Haemoglobin
polymorphisms. Hb
S. ·
Infected AS cells sickle more
readily than uninfected cells; this may lead to increased reticuloendothelial
clearance. ·
Main effect of HbS is strong
protection against the clinical effects of malaria. Infection is not markedly
less common in AS individuals Hb
C. ·
Cells of the homozygous individual
(CC) do not support parasite growth in culture due to resistance of red cells
bursting and releasing merozoites ·
AC cells support parasite growth
well ·
SC individuals common in areas of
high malarial endemicity ·
Parasite growth is poor in SC
cells Hb
E. ·
Parasitised EE and AE red cells
are phagocytosed more readily than AA red cells ·
Protection derives in part from
host immune system
Thalassaemia
& G6PD deficiency. The red cells of thalassaemia and G6PD deficiency may not support division of P. falciparum due to their sensitivity to oxidant stress. [Index] The biochemistry of Plasmodia. The metabolic requirements
of Plasmodia are obtained from the
haemoglobin of the host red cells and from the nutrients available from the
plasma. The presence of the
parasite in the red cell increases its permeability through the membrane for the
passage of nutrients, but the biochemical exchanges are very complex. Protein synthesis is
achieved from the essential amino acids, including cysteine and methionine,
present in the host plasma. The degradation of haemoglobin is incomplete,
leaving the malaria pigment haemozoin as a residue. Nucleic acids are partly
synthesised by the parasites and partly obtained by other pathways. A cofactor,
tetrahydrofolate (THF) is important for the conversion of certain amino acids
and for the synthesis of purine nucleotides; this cofactor is produced by
malaria parasites through a reaction in which an enzyme, tetrahydrofolate
reductase (THFR) is involved. It is this particular pathway that is the point of
action of some antimalarial drugs such as pyrimethamine, proguanil and
sulphonamides. The state of nutrition of
the host has an important bearing on the multiplication of plasmodia. It has
been found that para-amino-benzoic acid (PABA) must be present in the diet of
the host. PABA is one of the essential building blocks of the molecule folic
acid that is needed for the growth of mammalian plasmodia. Biochemical changes
produced by the aging of the red cells have an important bearing on their
susceptibility to invasion by plasmodia. P.
vivax and P. ovale are
predominantly found in young red cells. P.
malariae seems to prefer mature red cells while P.
falciparum appears to be indifferent to the age of its host cell. The receptor for merozoites
of P. falciparum is glycophorin, a
glyco-protein component of the red cell membrane. P. vivax uses the Duffy determinants for invasion of the red cells. Therefore
Duffy negative (Fya and Fyb) individuals are completely
resistant to infection with P. vivax - this
parasite is not endemic in West Africa, where the majority of people are Duffy
negative. The surface receptor molecules for P. ovale and P. malariae are as yet unknown. [Index]
Methodology. R66
formulation Giemsa diluted in pH 7.2 buffer is the stain of choice to optimally
demonstrate the microscopic intricacies of the malarial parasite. One
of the unfortunate drawbacks of Giemsa is that a certain amount of precipitate
is deposited on the film during the staining process. Filtration prior to use
does not overcome this problem. Once diluted in buffer the stain naturally
precipitates over a period of time. The WHO recommends that 200
fields must be examined before the film is declared negative. The time that this
takes will obviously vary from person to person. How long is a piece of
string? There are to my knowledge no WHO recommendations relating to this
problem. Laboratories who do not use thick films must rely on clinical
information, local staffing resources and their own conscience. There are several reasons
for this disastrous situation. Most common is the vigorous and enthusiastic
interpretation of the word ‘thick’. The thick film is made by using ~5μL
of blood, i.e. the same volume used for making thin films. Inadequate drying of
the film may hasten its journey down the sink! Depending on ambient temperature,
10-15 minutes should suffice. An excessive amount of EDTA will prevent good
adherence of blood to slide. Note that thick films from anaemic patients often
show some clumping of the red cells while the film is drying. During staining
aggregates of clumped red cells frequently wash off the slide resulting in
rather a mottled or patchy film. There are a variety of
immunochromatographic tests such as the ParaSight F™ and the ICT that
capture and detect the histidine rich protein 2 (HRP-2) antigen, and the OPTIMAL®
that detects Plasmodium lactate dehydrogenase (pLDH). Although the manufacturers
claim high levels of sensitivity and specificity, published trials have not been
quite so impressive. Their main use is for screening or confirmatory purposes. They
are not recommended as replacement methods for thick and thin films. Other techniques utilise
fluorochrome dyes such as acridine orange. Malaria parasites are easily
recognisable under UV light. This concept is used in the Beckton & Dickinson
QBC apparatus. PCR methodology is available but due to time constraints, it
cannot be regarded as a primary tool for the acute diagnosis of malaria. Microscopic appearance. No. These are parasite-induced
intraerythrocytic membranes thought to be associated with the transport of
proteins from the parasitophorous vacuole membrane to the host cell cytoplasm. These are caveolae-vesicle
complexes (small indentations in the red cell) which stain pink with Giemsa @ ph
7.2. If a more acidic buffer is used (pH 6.8) these stipples are not visible.
They are diagnostic of P. vivax or P. ovale. Schüffner’s dots
are finer than James’s dots. They are not uncommon.
Mixed infections may be missed if meticulous scanning of the thick film is not
carried out. Certainly. It is a rare
occurrence. The other problem is that
no two strains of the same parasite are identical; intra-species variation is a
characteristic of the malarial parasite. The parasites. How/why/when
did the Plasmodia species acquire their names? P. malariae
(Laveran 1881) – after the specific name of the organism P. vivax
(Grassi & Feletti 1890) – after the Latin 'vivax' - lively active
movement. P. falciparum
(Welch 1897) – after the Latin word ‘falx’ - a sickle (Shape of the
gametocytes) P. ovale
(Stephens 1922) – after the characteristic oval shape of red cells infected
with this parasite. Please note however that only ~30% of infected red cells
demonstrate the classical fimbriated oval shape. Geographical
distribution? Trivia – did you know that? 
William Shakespeare was well acquainted with malaria – ‘He is so shak’d
of aburning quotidian tertian that it is most lamentable to behold’. (Henry V, II)
[Index] Three species of the genus
Trypanosoma are responsible for disease in humans. Salivarian
trypanosomes. Trypanosoma
bruceii rhodesiense. Trypanosoma bruceii gambiense. The metacyclic trypanosomes
are in the proboscis of the insect vector - infection is therefore inoculative.
The above are the aetiological agents of African trypanosomiasis. Stercorarian
trypanosomes. Trypanosoma
cruzi. Trypanosoma rangelli. *
The metacyclic trypanosomes
occupy a posterior position in the gut of the insect vector and are passed out
in the faeces - infection is therefore contaminative. The above are the
aetiological agents of South American trypanosomiasis. * Only found in very rare
cases. Structure
of the parasite. The parasite is an
elongated cell with single nucleus that usually lies near the centre of the
cell. Each cell bears a single flagellum that appears to arise from a small
granule - the kinetoplast. The kinetoplast is a specialised part of the
mitochondria and contains DNA. The length and position of the trypanosome’s
flagellum is variable. In trypanosomes from the blood of a host the flagellum
originates near the posterior end of the cell and passes forward over the cell
surface, its sheath is expanded and forms a wavy flange called an undulating
membrane. Transmission
and vectors. Trypanosomes are
transmitted by blood sucking insects. The African trypanosomes are transmitted
by tsetse flies (Glossinia). Triatomid and Reduvid bugs transmit the South
American trypanosomes. In the insect gut, the trypanosomes multiply extensively and undergo morphological change. The completion of the cycle results in metacyclic trypanosomes being present either in the gut of the vector (South American species) or in the proboscis of the vector (African species)
Diagnosis of Trypanosomiasis. Laboratory diagnosis of
African trypanosomiasis is by; 1. Examination
of blood for the parasites 2. Examination
of aspirates from enlarged lymph glands for the parasites 3. Examination
of the csf for the parasite 4. Detection
of trypanosomal antibodies in the serum Examination
of blood. The
following techniques are recommended. 1. Thick blood film 2. Buffy coat
examination 3. Triple centrifugation
technique 4. QBC 5.
Miniature anion-exchange centrifugation technique (ref. Transactions Royal Society of Tropical Medicine and Hygiene. 1979. 73. 312-317 Examination
of lymph gland aspirates. The aspirate can be
examined microscopically by making a wet preparation, or if there is not much
material, it can be allowed to dry on a slide and then stained with either rapid
Field’s stain or with Giemsa. Examination
of csf. In the late stages of
African trypanosomiasis, trypanosomes may be found in the csf together with IgM
- containing morula (Mott) cells, lymphocytes and other mononuclear cells. Once
the csf has been collected it must be
examined as soon as possible. The parasites are unable to survive for more
than 15-20 minutes in csf once it has been removed. The parasites become
inactive, are rapidly lysed and will not therefore be detected. The csf should
be examined wet and spun down In a sterile tube and a film made from the
deposit. This is then stained with rapid Field’s or Giemsa. NB. it is impossible to speciate T. brucei var. gambiense from T. brucei var. rhodesiense on a stained film. [Index] The parasites of the genus
Leishmania form a large complex of organisms that may infect a large range of
vertebrates. All Leishmania species are
transmitted by the sandfly (Phlebotomus or Lutzomyia). A species of sandfly that
is a vector of one species of Leishmania may not be able to transmit another. Human Leishmaniasis can
manifest itself in three broad clinical forms.
Smears and cultures of the
material should be made. Smears
(marrow, splenic aspirate, slit
skin
). 1. Air dry smears. 2. Fix in methanol for 1
minute 3.
Stain with Giemsa 1 in 10 in buffered distilled water pH 6.8 for 30
minutes (or use the rapid Field’s stain) 4. Wash the slide in
buffered water and drain dry Amastigotes of leishmania
should be seen in positive smears. They are approximately 2-4 µm in size, oval
and are frequently seen within the cytoplasm of the macrophage. The amastigotes
possess a nucleus and a rod - shaped kinetoplast within the cytoplasm. In many samples, a very small number of parasites are present. Extensive searching of the film is necessary.
Amastigotes
of L. donovani. Splenic aspirate.
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