the 51 patients with kala azar, all had fever lasting from <1 month to 1.5 years (median, 4.5 months). All six PKDL patients gave a history of having kala azar in the past, and their slit skin test smears were microscopically positive for Leishman-Donovan bodies. The strip test was positive in all the cases of kala azar and PKDL (estimated sensitivity, 100%), and all control sera were negative by the ICT (specificity, 100%). The rK-39 ICT is a highly sensitive and specific test and may be suitable for a rapid, cost-effective, and reliable field diagnosis of kala azar and PKDL (55).
This test has also been adapted to the use of urine (56) and sputum (57) for the detection of visceral leishmaniasis antibody. Both screening approaches appear to be very promising, with distinct advantages over the invasive use of blood specimens.
Cytocentrifugation (cytospin), which uses an apparatus for concentrating cells in suspension on a microscope slide, is commonly used in most histopathology laboratories. The use of a hemolyzing and isotonic saponin solution to lyse RBCs and platelets, which contains formalin as a fixative, has led to an improved technique for the detection of Plasmodium spp., L. donovani, microfilariae, and WBCs containing malaria pigment. The concentration of the parasites present in the sediment from 100 µl of blood spread on a 6-mm-diameter circle results in good morphology that is well stained using Giemsa, Wright’s, or rapid stains. This new method costs very little to perform and offers the possibility of isolating and identifying the main blood-stage parasites in the same sediment. The possible exception would be young trophozoites of P. falciparum, which do not concentrate well due to their small size (58).
The Knott concentration procedure is used primarily to detect the presence of microfilariae in the blood, especially when a light infection is suspected (4, 59) (Fig. 7.15). The disadvantage of the procedure is that the microfilariae are killed by the formalin and are therefore not seen as motile organisms.
Figure 7.15 Microfilariae at low power from a Knott concentration, unstained. Note that the sheath is not visible. doi:10.1128/9781555819002.ch7.f15
Figure 7.16 African trypanosomes obtained using the triple centrifugation method. doi:10.1128/9781555819002.ch7.f16
1. Place 1 ml of whole or citrated blood into a centrifuge tube containing 10 ml of 2% formalin. Thoroughly mix the tube contents.
2. Centrifuge for 2 min at 500 × g or 5 min at 300 × g.
3. Decant the supernatant fluid, examine some sediment as a wet mount at low (×100) and high dry (×400) power, prepare thick films from the remaining sediment, and allow the films to air dry.
4. Stain films with Giemsa, Wright’s, or rapid stains.
Note Use alcohol-cleaned slides for preparation of the films made from the sediment.
The membrane filtration technique as modified by Desowitz and others has proved highly efficient in demonstrating filarial infections when microfilaremias are of low density. It has also been successfully used in field surveys (3, 4, 60, 61). (See also Fig. 5.9, which shows a membrane filtration system that can also be used for blood filtration; see below.)
1. Draw 1 ml of fresh whole blood or anticoagulated blood into a 15-ml syringe containing 10 ml of distilled water.
2. Gently shake the mixture for 2 to 3 min to ensure that all blood cells are lysed.
3. Place a 25-mm Nuclepore filter (5-µm porosity) over a moist 25-mm filter paper pad. (This method is unsatisfactory for the isolation of M. perstans microfilariae because of their small size. A 3-µm-pore-size filter could be used for recovery of this organism. Other filters of similar pore size are not as satisfactory as the Nuclepore filter.) Place the filter in a Swinney filter adapter.
4. Attach the Swinney filter adapter to the syringe containing the lysed blood.
5. With gentle but steady pressure on the piston, push the lysed blood through the filter.
6. Without disturbing the filter, remove the Swinney adapter from the syringe and draw approximately 10 ml of distilled water into the syringe. Replace the adapter, and gently push the water through the filter to wash the debris from the filter.
7. Remove the adapter again, draw the piston of the syringe to about half the length of the barrel, replace the adapter, and push the air in the barrel through the filter to expel excess water.
8. To prepare the filter for staining, remove the adapter, draw the piston about half the length of the barrel, and then draw 3 ml of absolute methanol into the syringe. Holding the syringe vertically, replace the adapter and push the methanol followed by the air through the filter to fix the microfilariae and expel the excess methanol.
9. To stain, remove the filter from the adapter, place it on a slide, and allow it to air dry thoroughly. Stain with Giemsa stain as for a thick film (with 0.1% Triton X-100) or with Delafield’s hematoxylin.
10. To cover the stained filter, dip the slide in toluene before mounting the filter with neutral mounting medium and a coverslip. This will lessen the formation of bubbles in or under the filter.
Gradient Centrifugation Technique
The gradient centrifugation technique is another technique for the concentration of microfilariae (62).
1. Mix 30 ml of 50% Hypaque with 14 ml of distilled water; add 1 part of this mixture to 2.4 parts of 9% Ficoll.
2. Place 4 ml of the Ficoll-Hypaque mixture in a 15-ml plastic centrifuge tube; overlay this mixture with 4 ml of heparinized venous blood.
3. Centrifuge the tube at 400 × g for 40 min.
4. Microfilariae will be found in the middle Ficoll-Hypaque layer, which separates the overlying plasma and WBC layers from the underlying RBCs.
