Blood parasites
Written by Tim Inglis


Blood parasites

Lynne Garcia, LSG & Associates, Santa Monica, CA., USA.

Parasitology Masterclass, RMIT, Melbourne, 14th August, 2010.

41% world’s population is exposed to endemic malaria. 2.7 million die from malaria each year. Malaria was eradicated in Australia as recently as 1981.  Mosquito bite is the main method on transmission, but there are several other means of infection.


Life cycle. The early stages are not synchronised and lack the periodicity due to synchronised parasite release into the bloodstream. Laboratory diagnosis is difficult when parasitaemia is low. WHO defines any malaria parasite that originates from the liver again as a relapse. Emergence of gametocytes takes 1-2 weeks. Patients in the emergency department suspected of having malaria need blood drawing immediately, rather than waiting for the perfect timing. The need for a 24/7 service means that in many places malaria screening is done by Haematology rather than Microbiology. Whoever does them, good blood smears are an art and should be made as soon as the blood has been drawn. EDTA blood is better than heparinised blood.


CAP standard required both thick and thin films to be examined before a report can be issued. False negative smears can be caused by low parasitaemia, particularly after commencing treatment. So a travel history is mandatory. Malaria films should be a STAT preparation, the only other truly emergency parasitology being CSF examination for amoebae. Thick and thin films need 300 high power fields examined per film (i.e. x 1000 magnification)


Quality control for malaria films. This is now more practical and does not depend on sending out samples for review. The current approach is built into the system in use and judged by how good the leukocytes appear.


Variation & distortion of parasites. Beware real life variation e.g. P falciparum rings are often bigger then the textbook appearance. Distortion begins around 1hr after blood has been drawn. Typical appearance can be lost by 4-6hr. Gametocytes may round up and become unrecognisable, even exflagellating. Refrigeration of EDTA specimen is not recommended because parasites round up.


Stains for blood films. Giemsa is the stain of choice but may be difficult or costly. There are now many proprietary options e.g. DipStat stain. While colour is not critical to detection, the appearance of leukocytes is important (see QC comment above). There are pros and cons for both thick and thin films. Thick film is much more sensitive, picking up more infections than thin film but is more difficult to interpret and to use as means of species identification. A Coplin jar should not be used for stains. A quick double-dip in acetone once the blood smear has dried can produce a clearer film. A new has been developed technique for combining thick and thin smears.


Rapid tests.

Buffy coat smears. These can be more sensitive but P falciparum rings are less buoyant. The proprietary QBC (acridine orange) tubes are suitable for the classic stages.


A thick film will be positive at around 0.0001% or 5-20 parasites per uL, while a Binax Now card test will decline in sensitivity below 0.1% i.e. not better than a trained microscopist. External controls are needed but the result cannot be reported without performing films. These are quite good for P falciparum and P vivax but only at high level parasitaemia. There is a risk of false negative results if reliance is placed on a rapid test only. Failure to detect Plasmodium sp may result in patient death.



  • Oil on microscope slides
  • Need for standard precautions for occupational health & safety
  •  Letting blood sample stand too long before preparing films
  •  Sending report out without examination of a blood film


Species-specific notes (recognition chart) (CDC guide):


P vivax. Appearance is not always typical. There may be a steady low-grade fever in early infection.

P ovale. This infects young erythrocytes and can be easily confused with P vivax infection. The trophozoites moves more slowly than does P vivax and leaves a fimbriated edge to the infected RBC. The world distribution of P ovale infection is restricted.

P malariae. Band forms are characteristic but not limited to P malariae. Infection may remain for 20-40 years. Infection produces lots of malaria pigment and immune complex with time, contributing to kidney damage, leading to nephritic syndrome. The schizont is daisy head shape.

P falciparum. This causes most of the severe cases of infection, the majority of infections in the USA and produces greatest scope for failures. Mixed infections are easily missed. The early stages of infection don’t necessarily resemble malaria. Microscopic features that help distinguish this from other Plasmodium spp include accole and appliqué forms, gametocyte shape, ingestion of malaria pigment, double chromatin dots and exflagellation of gametocytes.

P knowlesi. This recently recognised Plasmodium infection of humans also causes infection of long tailed macaques and is found in Sarawak, Singapore, the Philippines, and along the Thai-Myanmar border. There is a 24hr cycle affecting all ages of erythrocyte. Rings can be delicate and Schuffner’s dots missing so that microscopic features of early infection resemble P falciparum, but later stages can mimic P malariae. 10% human infections are fatal and the severe complications resemble P falciparum. Rapid methods do not identify to species level.

Mixed infections. These are underestimated. E.g 30% may be mixed P falciparum/P malariae in Thailand. It appears that Anopheles species can transmit two Plasmodium species at one time.


Other blood parasites.


Babesiosis. Most often B.microti, mimics P falciparum with many ring forms on the blood film but no other stages. The characteristic Maltese Cross formation may not be visible on blood film. Lack of familiarity can cause long delays in arriving at a definitive diagnosis. This is a tick-borne infection in Europe and the USA, particularly in asplenic patients.  Disease on the West Coast of the USA is more severe. Immediate thick and thin films need performing on patients with suspected Babesiosis. Babesia can be difficult to see on buffy coat smears by QBC and the ring forms can be outside RBCs.


Leishmaniasis. There are over 2 million cases of visceral and cutaneous leishmaniasis per annum worldwide. CL is now endemic in Texas. Clinical disease covers a spectrum of visceral, mucocutaneous and cutaneous. Following the first Gulf War there were 20 cases of CL caused by L major and 12 cases of VL caused by L tropica in returning troops In a survey from 2002-2004, over 500 cases of CL were detected due to L major in personnel returning from Iraq. It took up to around 60 days to develop skin lesions at up to 9 locations. Some showed evidence of reinoculation at multiple points consistent with slipping bandages. Bed nets and permethrin treatment of uniforms were used as control measures. Leishmania culture needs a positive control. True amastigote stage appearance can be seen when parasite released from macrophages on tissue squash prep. Samples best obtained by punch biopsy around the edge of the lesion after it has been carefully cleaned to avoid bacterial contamination of culture medium. A minimum of 3 samples is needed. The fine needle aspirate method has poor sensitivity. Culture works well on NNN or Schneider’s drosophila media. Best sensitivity is with real time PCR assay, where available. Patients probably have infection for life despite treatment, options for which include doing nothing, heat, cryotherapy, topical Paromomycin and pentostam. MCL in Mexico, Belize, Guatemala and the Southern USA due to L mexicana. VL associated with rise in immunoglobulins but antibodies do not eradicate infection. Recovery due to treatment may be accompanied by development of post kala-azar dermal Leishmaniasis.


Trypanosomiasis. American and African forms. The gambiense form of African trypanosomiasis is particularly difficult to diagnose. The American form (Trypanosoma cruzi) has caused infection in around 150,000 people, particularly in family donors in Teas & California. The Triatomid bugs leave an allergen causing scratching which leads to inoculation of parasite in bug faeces. Transmission also via intravenous drug use. Infection results in myocarditis and megacolon. 

Notes by TJJI, AUG-2010