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Keywords:

  • microfilaria;
  • concentration technique;
  • karyotype;
  • bone marrow;
  • peripheral blood
  • microfilaires;
  • technique de concentration;
  • caryotype;
  • moelle osseuse;
  • sang périphérique
  • Microfilaria;
  • técnica de concentración;
  • cariotipe;
  • médula ósea;
  • sangre periférica

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

During karyotype preparation from the bone marrow aspirates of 209 haematological malignancy cases, microfilaria were detected in four samples, whereas routine marrow and peripheral blood smears of these four cases did not show any parasite. The patients were recalled, and their peripheral blood was processed by karyotyping and standard concentration techniques. Karyotype preparation from peripheral blood was performed with and without addition of colchicine. When the blood was processed for karyotyping with colchicine, microfilaria were detected in the peripheral blood of all four patients. In samples without added colchicine, no parasite was observed. The same samples were processed by Knott’s concentration technique, which showed microfilariae only in one of the four patients. Routine thick and thin smears of these patients showed no parasite. It seems that the standard karyotype preparation technique with colchicine concentrates the microfilariae in samples where parasite load is small and not demonstrable with standard techniques. Serological tests are available for W. bancrofti and costly, whereas no regular serodiagnosis is available for B. malayi. In a country like India, both parasites are endemic and patients are treated on clinical suspicion when parasitaemia could be low. Low parasitaemia is common because of repeated infection and partial immunity. In such circumstances, a cost-effective concentration technique like this may be useful.

Lors de l’établissement du caryotype de ponctions de moelle osseuse de 209 cas d’hémopathie maligne, les microfilaires ont été détectées dans 4 échantillons, alors que les frottis de routine de la moelle et du sang périphérique de ces 4 cas n’ont pas révélé de parasite. Les patients ont été rappelés et leur sang périphérique a été traité pour le caryotypage et par les techniques de concentration standard. La préparation pour le caryotypage du sang périphérique a été réalisée avec et sans addition de la colchicine. Lorsque le sang a été traité avec de la colchicine pour le caryotypage, les microfilaires ont été détectées dans le sang périphérique de l’ensemble des 4 patients. Dans les échantillons préparés sans colchicine aucun parasite n’a été observé. Les mêmes échantillons ont été traités par la technique de concentration Knott, qui a révélé des microfilaires chez un seul des 4 patients. Les frottis minces et épais de routine de ces patients n’ont révélé aucun parasite. Il semble que la technique de préparation pour le caryotypage standard avec de la colchicine concentre les microfilaires dans les échantillons où la charge parasitaire est faible et non détectable avec des techniques standard. Des tests sérologiques sont disponibles pour W. bancrofti et sont coûteux, alors qu’aucun diagnostique sérologique courant n’est disponible pour B. malayi. Dans un pays comme l’Inde, les deux parasites sont endémiques et les patients sont traités sur base de la suspicion clinique lorsque la parasitémie paraît faible. La faible parasitémie est fréquente due à une infection répétée et une immunité partielle. Dans de telles circonstances une technique de concentration rentable telle que celle-ci peut être utile.

Durante la preparación de cariotipos a partir de los aspirados de médula ósea de 209 casos de cáncer hematológico, se detectaron microfilarias en 4 muestras, mientras que las pruebas rutinarias de médula ósea y frotis de sangre periférica de estos 4 casos no mostraban ningún parásito. Se citó de nuevo a los pacientes y se procesó su sangre periférica para cariotipado y técnicas de concentración estándar. La preparación de los cariotipos a partir de sangre periférica se realizó con y sin añadir colchicina. Cuando se procesó la sangre para el cariotipado con colchicina, se detectaron microfilarias en la sangre periférica de los cuatro pacientes. En muestras sin añadir colchicina no se observaron parásitos. Las muestras se procesaron mediante la técnica de concentración de Knott, observándose microfilarias en uno de los 4 pacientes. En los frotis de sangre gruesa y delgada realizados de rutina a estos pacientes no se observaron parásitos. Parece que la técnica de preparación estándar de cariotipos con colchicina concentran las microfilarias en muestras en las que la carga parasitaria es pequeña y no demostrable mediante técnicas de diagnóstico estándar. Hay pruebas serológicas disponibles para W. bancrofti y son costosas, mientras que no hay un serodiagnóstico regular para B. malayi. En un país como la India, ambos parásitos son endémicos y los pacientes son tratados por una sospecha clínica cuando la parasitemia podría ser baja. La baja parasitemia es común debida a la infección repetitiva que provoca una inmunidad parcial. Bajo estas circunstancias, disponer de una técnica barata para concentrar la muestra, como la aquí descrita, sería muy útil.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Filarial infection is an important cause of morbidity in many coastal states of India, where two types of filarial infection caused by W. bancrofti and B. malayi occur. Detection of microfilaria in peripheral blood is important. In addition to simple thick and thin blood smear tests, concentration techniques are used: mainly density gradient centrifugation, haemolysis and filtration of the blood through a polycarbonate membrane, which retains the parasite (Denham 1975; Jones et al. 1975; Feldmeier et al. 1981). Knott’s technique, one of the simplest known, uses 2% formalin in distilled water to concentrate the parasite. Diethyl carbamazine provocation and the diurnal rhythm of the parasite followed by timed blood sample collection have been combined with concentrating techniques to improve the positive yield of microfilaria detection. Recently, serodiagnosis of W. bancrofti provides a simple but costly means of diagnosis (Weil et al. 1997). In W.bancrofti and B. malayi endemic areas, serodiagnosis may not be superior to the detection of microfilaria in the circulation for active infection. We found that a standard karyotyping technique seems to improve the positive yield of microfilaria.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

For peripheral blood smears, thick and thin smears were prepared from EDTA blood samples directly, or from the water-lysed blood sample and observed under the microscope for microfilaria. For Knott’s technique, 1 ml of blood was lysed and concentrated using 2% formalin (Denham 1975).

For the karyotype technique, 1 ml of heparinised bone marrow was incubated with 5 ml of complete media and colchicine in a centrifuge tube for 30–60 min at 37 °C. After incubation, the sample was centrifuged for 10 min and the supernatant was discarded. 5–7 ml of hypotonic solution (0.75 m KCl) was added and incubated for 30 min at 37 °C. The sample was centrifuged and the supernatant discarded. The cells were fixed with 3–5 ml of Carnoy’s fixative, mixed well and centrifuged. This step was repeated 3–4 times until the pellet became white. Slides were prepared by dropping the cell suspension on a wet slide. The slides were air-dried and aged at room temperature for 4–6 days. GTG banding was one and observed under the light microscope (Seabright 1971).

To use karyotyping for microfilaria detection, 2 ml of blood sample in two centrifuge tubes (1 ml in each tube) was processed according to the cytogenetic technique with and without using colchicine to detect the microfilaria, omitting the binding step. The slides were stained directly with Giemsa stain and observed under the microscope.

In another set of investigations, six blood samples with high microfilarial count (as shown by counting the microfilaria in counting chamber) were diluted with group compatible afilariamic blood obtained from a blood bank and the infected samples were diluted serially to 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:240. The diluted samples were processed with all six techniques, and microfilaria were demonstrated on a slide. The highest dilution in which microfilaria detected was observed under low power field (10×) by all investigators to verify the sensitivity of the technique. Microfilaria demonstrated by any one investigator were taken as positive, and the result was converted into microfilarial density which could be visible by standard light microscopy with the given technique, that is, if original microfilaria density was 400 ml and the microfilaria was seen up to 1:40 dilution, then sensitivity of the technique was 10 Mf/ml.

A literature search for comparative evaluations of various techniques of microfilaria demonstration rendered the studies presented in Table 3.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Microfilaria were demonstrated in peripheral blood using karyotyping with colchicine in four of 209 patients and in one of 209 samples using Knott’s technique. Standard karyotyping with colchicine revealed parasites in all cases, but without colchicine in none (Table 1). Under low power (×10) magnification, 2–5 parasites were seen in the karyotype preparation of the marrow (Figure 1) and identified as W. bancrofti.

Table 1.   Microfilaria detected in peripheral blood from four patients described in Table 1 by different techniques seen under ×10 objective
Sr.NoThick & thin smear Mf/10 LpfW/L thick smear Mf/10 LpfW/L thin smear Mf/10 LpfKnott’s technique Mf/10 LpfK/P with Colchicine Mf/10 LpfK/P without Colchicine Mf/10 Lpf
  1. W/L, Water-Lysed smear; K/P, Karyotype Processed; Mf/10 Lpf, Average no of Microfilaria seen in 10 low power field (Lpf).

1000020
2000020
3000030
4010310
image

Figure 1.  Slide showing 5 Microfilariae in cytogenetic preparation. (a) Microfilaria under 100×. (b) Metaphase under 100×.

Download figure to PowerPoint

Table 2 shows another set of investigations that determined the absolute concentration of microfilaria by Knott’s technique and karyotype processing with and without colchicine. With Knott’s technique, we could detect up to 23 Mf/ml in contrast to karyotype processing with colchicine, which detected a microfilarial density as low as 9 Mf/ml. The karyotype procedure without colchicine was slightly inferior to Knott’s technique, showing that colchicine is an important component of the process where parasite concentration was 34-fold (Table 3) (P = 0.0015 Knott’s technique v/s karyotype with colchicine).

Table 2.   Threshold density at which microfilaria was detected by different techniques
No. of sampleCounting chamber method mf/ml bloodKnott’s method mf/ml in (highest dilution)KP with colchicines mf/ml in (highest dilution)KP without colchicine mf/ml in (highest dilution)
  1. Paired t test: Knott’s test v/s K.P with Colchicine P = 0.0015.

  2. With colchicine vs without colchicine P = 0.0059.

  3. M, mean.

1371  18 (1:20) 9 (1:40)  18 (1:20)
2431  22 (1:20)11 (1:40)  44 (1:10)
3272  27 (1:10) 7 (1:40)  27 (1:10)
4400  20 (1:20)10 (1:40)  20 (1:20)
5140  28 (1:5) 7 (1:20)  28 (1:5)
6221  22 (1:10)11 (1:20)  22 (1:10)
 183513755159
  M = 306 M = 23 (×14) M = 9 (×34) M = 27 (×12)
Table 3.   Different techniques used for microfilarial detection
Sr No.Name of the techniquesSensitivityReferences
 1Standard thin smearNot very sensitive (20%) Goldsmid (1970)
 2Standard thick smearMore sensitive than thin smear (50%) Goldsmid (1970)
 3Wet preparationMoving mf can be observed and more sensitive than above (60%) Goldsmid (1970)
 4Polyvidon techniqueMore sensitive than above (65%) Goldsmid (1970)
 5Microhaematocrit techniqueMore sensitive than above 4 methods (90%) Goldsmid (1970)
 6Denhams counting chamber method25–35% higher than the thick smear method Denham (1975)
 7Membrane filtration tech.Nucleopore method is approximately 35 times as good as the Millipore method and superior than Knott’s techniqueAnnals of Tropical Medicine and parasitology, 61,220–223
 8Finger prick blood filmVery low sensitivity.Transactions of the Royal Society of Tropical Medicine and Hygiene (1991) 85,506–510
 9Modified MF and concentration method.Very highly sensitive than thick film method.Journal of Parasitology (1970), 56,623–624
10Filarial antibodies testHighly sensitive technique Wong (1969)
11Use of larger volume of blood in low density of mfSensitivity is moderate but impractical for small childrenS.E. Asian F. trop.Med.publ,Hlth, (1973),4, 172–75.
12Density gradient method using Percoll-solutionTest is simple and sensitive in low microfilaraemiaFeldmeier (1981)
13Concentration techniques Using 2% saponinMore sensitive than Knott’s methodJournal of tropical medicine and hygiene. 1990, 93, 390–392.
1410% Teepol, membrane filtration concentration techniqueBoth saponin and Teepol membrane filtration method are more sensitive than Knott’s method Dickerson (1990)
15Knott’s technique using 2% formalinThis method does not detect all the microfilaria in the sampleAnn. Soc.belge Med.trop.1975, 55, 5, 517–524
16Sasa’s concentration techniqueMore sensitive than Knott’s methodAnn. Soc.belge Med.trop. 1975, 55, 5, 517–524
17Present technique (cytogenetic technique, a concentration method)Appears to be as sensitive as filtration technique 

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Bancroftian filariasis is widely distributed and is responsible for 98% of filarial infection in India. Maharashtra, where Mumbai city is situated, has microfilaria prevalence rate of 7.27% (Sabesan et al. 2000). Brugian filariasis has a localised and restricted distribution in India comprising only 2% (Sabesan et al. 2010) and occurs mainly in the central part of Kerala State along the coast. Smaller pockets of B. malayi infection exist in Andhra Pradesh, Tamil Nadu, Assam, Orissa and Madhya Pradesh (Russel et al. 1979).

In endemic areas, microfilaria can be detected in bone marrow (Rani & Beohar 1981; Hemachandran et al. 2003) and in many other pathologic samples (Singh et al. 2000; Kishore et al. 2008; Maheshwari et al. 2008). Whether microfilariae in these cases are pathologically related to underlying disease (Sharma et al. 2006) or mere passengers is debatable (Bagla et al. 2005).

In the present study, microfilaria were seen in approximation 2% of 209 marrow aspirates (209) sent to us for karyotyping because the underlying disease in all these patients was either a haematological malignancy or myelodysplastic syndrome. Two of our patients with microfilaria in the marrow had multiple myeloma, and the other two had acute lymphoblastic leukaemia and chronic myeloid leukaemia. It is not out of place to mention that microfilaria has been demonstrated in a patient with Waldenstrom’s microglobulinaemia (Mc Farlane & Nwokoto 1966) and rheumatoid arthritis. In the present study, it was possible to show that routine karyotyping with colchicine tends to concentrate the microfilaria. Colchicine is an important component of the process, as without it no parasites were seen. Even with standard concentrating techniques, parasite could only be seen in one case. Whether concentration of the parasite using filtration would have shown parasitaemia in these four cases remains unclear. To better understand this, samples with high levels of parasites were studied quantitatively after serial dilutions by different techniques. The degree of concentration achieved by different techniques (Table 2) clearly shows that a higher level of parasite concentration was achieved by karyotyping with colchicine.

There are a number of tests and techniques available in the literatures to demonstrate microfilaria or microfilarial antigen in peripheral blood (Table 3). In addition, many tests employed diurnal rhythmicity or diethyl carbamazine provocation to improve the parasite density in peripheral blood. While there is no doubt that membrane filtration is the best technique for parasite demonstration in low parasitaemia, ours did not fare too badly. It remains to be seen whether the present test can be validated independently by others workers, while we continue to investigate the possible mechanism by which colchicine improves parasite detection.

Colchicine, a mitotic spindle poison, is widely used as an experimental tool to study cell division and microtubule and/or spindle function. It can arrest plant and animal cell division in vitro and in vivo. Mitosis is arrested in metaphase owing to failure of spindle formation. Colchicine inhibits the release of histamine-containing granules from mast cells, the secretion of insulin from beta cells of pancreatic islets and the movement of melanin granules in melanophores. Colchicine also exhibits a variety of pharmacological effects. It lowers body temperature, increases the sensitivity to central depressants, depresses the respiratory centre, enhances the response to sympathomimetic agents, constricts blood vessels and induces hypertension by central vasomotor stimulation. It enhances gastrointestinal activity by neurogenic stimulation but depresses it by a direct effect and alerts neuromuscular function (Insel 1997). The precise function of colchicine in microfilariae concentration is not known. However, there could be several reasons why addition of colchicine in basic karyotyping provided significantly better concentration of parasites (34 times with colchicines, 12 times without colchicines, p = 0.0059). Studies have shown that microfilaria tend to move with the leucocyte layer when concentrated by Percoll gradient (Feldmeier et al. 1981). Hence, when whole blood is centrifuged, we may expect the parasite to be associated with buffy coat layer. Addition of colchicine kills the organism and allows it to stay in the buffycoat layer rather than moving away from it. Colchicine also probably increases the adherence of leucocyte to the parasite (Figure 1a) making it heavier. During subsequent hypotonic lysis, the parasite treated with colchicine might imbibe more water than a non-treated parasite, making it even heavier – in contrast to Knott’s technique, where the added formalin not only kills the parasite but also fixes it and renders it impermeable to water. In the present technique, fixation comes much later and Carnoy’s fixative does not dehydrate the parasite. Combination of hydrated parasite with attached leucocytes makes the parasites quite heavy, and in the presence of light density of Carnoy’s fixative (Glacial acetic acid+ methanol), the parasite quickly settles at the bottom of the tube during subsequent centrifugation. The deposit so examined had concentrated the parasite more than 30-fold. Colchicine, Carnoy’s fixative and hypotonic solution are not costly reagents. Hence, the present technique is cheap and can be used in any modest parasitology laboratory. Present study may be considered a preliminary study. More studies comparing different concentration techniques with the present technique using artificially prepared blood sample with graded numbers of microfilariae added may exactly pinpoint the threshold number of microfilariae, which could be detected from peripheral blood.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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