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Clinical diagnosis
It has to be mentioned that in many parts of the world, particularly in hyperendemic and holoendemic areas, physicians often go with presumptive malaria diagnosis based exclusively on the clinical symptoms. Various clinical algorithms have been suggested and the best of them predict only up to 50% of true malaria cases. This method has poor specificity and positive predictive value. It does not allow differentiation in between falciparum and vivax malaria. Moreover, many symptoms of presumptive malaria overlap with other diseases such as pneumonia, otitis, tonsillitis etc.

Blood smear examination
Today microscopic examination of the blood smear still remains the gold standard in malaria diagnosis despite the fact that many other techniques have become available. The aim of blood smear evaluation is to find out: 1. if the patient is infected 2. the type of plasmodium and life cycle stage 3. the level of parasitemia (number of parasites per 無 of blood). When performed correctly, this technique has a remarkable sensitivity. The parasite detection threshold for thin blood film is 100 parasites/無. For thick film the threshold is lower - about 5-20 parasites/無. In both cases the accuracy depends greatly on the experience of the microscopist. Overall, blood smear examination technique is simple, inexpensive but yet labor-intensive. It normally takes quite a time to examine 100 microscopic fields during each examination. It also requires appropriate equipment, reagents and trained personnel. Detailed guidelines for malaria specimen collection and blood smear preparation are provided by both WHO and CDC and may be downloaded from our web sites. Information on individual appearance of various stages of plasmodium species is also available ([link] to the separate .doc files on species under the microscope). Every clinic in the world has its own specifics in preparing malaria blood smear, yet there are general principles to follow. As a general rule, malaria blood film should be prepared as soon as possible after collecting venous blood. That will secure against the possible changes in parasite morphology and will also ensure the good staining characteristics. Particular attention should be given to the fixing stage of film preparation. Accidental fixing of the thick smear with methanol should be avoided, especially when both thick and thin blood smears are on the same slide. The examiner should let alcohol on patient's finger to dry before starting to prepare the thick smear. Enough care should be also taken to avoid fixing the thick smear while drying up the slide with heat.

So, the thin smear is fixed in methanol before staining; the thick smear is stained unfixed. Many hospitals have a Wright-Giemsa stain available, which is acceptable; however, Wright stain alone will not reliably stain Plasmodium parasites. For best results, the smear should be stained with a 3% Giemsa solution (pH of 7.2) for 30-45 minutes (see details in the link).

The correct thickness of the thick film can be judged by the legibility of the printed text seen through the slide. The print should be just legible.
In resource limited settings, thin and thick films may be taken on the same slide. The important hometake message is that both thick and thin smears should be always prepared. Both of them provide important information for diagnostic purposes (Table1).

Table1. Characteristics of thick and thin blood smears
Thick Smear Thin Smear
Lysed RBCs Fixed RBCs
Many layers Single layer
Large volume Smaller volume
Good screening test Good species differentiation
Low density infection can be detected Low density infection can be missed
More difficult to diagnose species Requires more time to read

The tables below can be used as a reference for both thin and thick film species differentiation (Tables2-3.).

Table2. Species differentiation on thin films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC   + +  
Infected RBC shape round round, distorted oval, fimbriated round
Stippling infected RBC Mauer clefts Schuffner spots Schuffner spots none
Trophozoite shape small ring, applique large ring, amoeboid large ring, compact small ring, compact
Chromatin dot often double single large single
Mature schizont rare, 12-30 merozoites 12-24 merozoites 4-12 merozoites 6-12 merzoites
Gametocyte crescent shape large, round large, round compact, round


Table3. Species differentiation on thick films
Feature P. falciparum P. vivax P. ovale P. malariae
Uniform trophozoites +      
Fragmented trophozoites   ++ +  
Compact trophozoites     + +
Pigmented trophozoites       +
Irregular cytoplasm   + +  
Stippling ("RBC ghosts")   + +  
Schizonts visible very rarely often often often
Gametocytes visible occasionally usually usually usually

The determination of the number of circulating parasites is exceedingly important for clinical purposes to monitor the evolution of the disease and the efficacy of therapy. Different methods have been proposed:

  1. Number of parasites/無 of blood (thick film): This method requires observation of as many microscopic fields (100x oil immersion lenses) necessary to count 200 white blood cells (WBC). Number of asexual parasites and WBCs should be counted in each field until the number of WBCs reaches 200.
    If number of WBCs is unknown, it can be assumed to be 8000//µL
  2. Number of parasites/無 of blood (thin film): This method requires the preliminary determination of the number of erythrocytes (RBCs) present in the average microscopic field.
    The number of asexual parasites is counted in at least 25 microscopic fields. The number of RBCs in the average microscopic field is about 200, so total RBCs counted in 25 fields is about 200*25 = 5000. If the hemogram is not available, RBCs/µL is assumed 5.000.000 for males and 4.500.000 for females.
  3. Proportion of parasitized erythrocytes/total RBC count (thin film): This method will indicate the percentage of erythrocytes that are infected by malaria parasites.
    The number of parasitized erythrocytes (asexual forms) present in 25 microscopic fields is counted divided by the total number of erythrocytes present in these fields (about 5000), and multiplied by 100.
  4. Semi quantitative count (thick film): This alternative method of estimating parasite density is very quick but less accurate. It should be used only when it is not possible to perform more accurate methods. The following semi-quantitative scale is used.
    +   1-10 asexual parasites per 100 thick film fields
    ++   11-100 asexual parasites per 100 thick film fields
    +++   1-10 asexual parasites per single thick film field
    ++++   > 10 asexual parasites per single thick film field

It is important to know that parasite blood density is correlated with the severity of clinical presentation (Table4.).

Table4. Level of parasitemia and clinical correlates
Parasitemia Parasites / l Remarks
0.0001-0.0004% 5-20 Sensitivity of thick blood film
0.002% 100 Patients may have symptoms below this level, where malaria is seasonal
0.2% 10,000 Level above which immunes show symptoms
2% 100,000 Maximum parasitemia of P.vivax. and P.ovale
2-5% 100,000-250,000 Hyperparasitemia/severe malaria*, increased mortality
10% 500,000 Exchange transfusion may be considered/high mortality
* WHO criteria for severe malaria are parasitemia > 10,000 / l and severe anemia (Hb < 5 g/l). Prognosis is poor if > 20% parasites are pigment containing trophozoites and schizonts and/or if > 5% of neutrophils contain visible pigment. Hanscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab. Haem. 21, 235-245.

Fluorescent microscopy
This method of malaria diagnosis is based on the ability of fluorescent dyes to detect RNA and DNA contained in parasites. As a reminder, there is normally no nucleic material in mature erythrocytes. Therefore, anything detected by the fluorescent die is assumed to indicate the presence of plasmodium. Various staining techniques have been designed to improve upon the examination of blood films. Kawamoto's technique assumes staining thin film with acridine orange (AO). This method is relatively cheap and highly sensitive (up to 90%). However, it requires sophisticated equipment (fluorescent microscope).
Quantitative Buffy Coat Assay (QBC) is another modification of fluorescent microscopy. Prior to the microscopy, the blood is centrifuged in AO-coated tubes. Both parasites and granulocytes take the dye. However, on the fluorescent microscopy, the parasites concentrate below the granulocyte level in tube. QBC method is quick and useful for screening large number of samples. However, it requires centrifuge and special stains along with fluorescent microscope. All of them have high cost. Moreover, the identification and quantification of species is difficult.

Serology
Serologic methods are based on the detection of antibodies against malaria parasites. Positive test can not distinguish between past or present infection and therefore it has limited value for treatment decisions. On the other hand, negative test may help to eliminate the possibility of malaria, because it has been shown that antibodies to asexual parasites appear some days after invasion of erythrocytes. The main use of serological tests is for:

  1. Retrospective confirmation of empirically treated non-immunes
  2. Tracing asymptomatic infections in blood donors
  3. Investigating congenital malaria especially when mother's blood smear is negative
  4. Epidemiological studies to define malaria transmission areas and monitor effectiveness of preventive intervention strategies

Malaria antigen detection
This method of malaria diagnosis is based on the number of immunologic assays to detect specific antigens. Only assays based on the detection of histidine-rich protein-2 (HRP-2) and the parasite lactate dehydrogenase (pLDH) have been commercialized so far. In both cases immunochromatographic rapid diagnostic test (RDT) is used with blood.
These easy to perform 'dipstick' tests have been developed to diagnose falciparum malaria rapidly. The table below summarizes the features of 2 major antigenic methods used today (Table5.).


Detection of malaria antigens with HPR-2 (A,C) and pLDH (B)

Table5. Rapid malaria detection tests
Feature PfHRP-2 tests pLDH tests
Test principle
  • Use of monoclonal antibodies
  • Detects a histidine-rich protein of P.falciparum
  • Water-soluble protein is released from parasitized RBCs
  • Not present in mature gametocytes
  • Use of monoclonal and polyclonal Ab
  • Detects a parasite enzyme, lactate dehydrogenase (pLDH)
  • pLDH is found in sexual and asexual forms
  • Differentiation between malarial species is based on antigenic differences between pLDH isoforms
Advantages
  • Threshold for parasite detection as low as 10 parasites/痞 (but sensitivity drops at < 100 parasites /痞)
  • Does not cross react with other species - P.vivax, P.ovale, P.malariae
  • Threshold for parasite detection ? 100 parasites/痞
  • Can detect all species which infect humans
  • Can differentiate between P.falciparum and non-falciparum malaria
  • Does not cross react with human LDH
  • Positive only in viable parasites, potentially useful for monitoring success of treatment
Disadvantages
  • Some tests only detect P.falciparum
  • Cannot detect mixed infections
  • Sensitivity and specificity decreases at < 100 parasites/痞
  • Can remain positive up to 14 days post treatment, in spite of asexual and sexual parasite clearance, due to circulating antigens
  • Cannot differentiate between non-falciparum species
  • Cannot detect mixed infections
  • Sensitivity and specificity decreases at < 100 parasites/痞
Sensitivity/Specificity* Sensitivity 92-100%
Specificity 85- 100%
Sensitivity P.falciparum 88-98%
P.vivax 89-94%
Specificity P.falciparum 93-99%
P.vivax 99-100%
Commercial cost/test** Approximately US$ 0.60 -1.00 Approximately US$ 2.50
Commercial products
  • PATH falciparum Malaria IC Strip test - Program for Appropriate Technology in Health
  • MAKROmed TM
  • Orchid ®
  • OptiMAL ® - Flow, Inc.
  • Binax NOW ® ICT Malaria - Binax, Inc.
* Compared to golden standard (microscopy)
**Varies by size of order and vendor

Polymerase chain reaction (PCR)
This method is based on the molecular biology technique to identify parasite genetic material. It has an extremely high sensitivity. The amplification principle behind the test allows to pick up a negligible amount of parasite DNA sequence and multiply it million times for easy detection. The detection threshold is as low as 0.05-0.1 parasite/µL. Today this method allows also a species differentiation. Moreover, it can be used to identify mutation, which can be correlated to drug resistance acquired by the parasite. However, the use of this promising technique is limited today to the large research labs. This method involves a high cost as well as trained personnel. The possibility of contamination (false positive) of the blood product remains another drawback of PCR.

 

© 2002. Malaria in Armenia.
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