HUMAN TRYPANOSOMOSIS

 

TRYPANOSOMOSE HUMAINE

 

Development of a direct immunofluorescence test (DIFAT) for parasitological diagnosis of African trypanosomOsis

 

Développement d’un test d’immunofluorescence directe (DIFAT) pour le diagnostic parasitologique de la trypanosomiase Africaine

 

1Lejon Veerle, 3Ameel Valéry, 2Brandt Jef, 2Van den Bossche Peter and  1Büscher Philippe

 

1Parasitology and 2Veterinary Departments, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerpen, Belgium.  Tel: +32 3 247 63 69, Fax: +32 3 247 63 73, Email: vlejon@itg.be,  3Provinciale Industriële Hogeschool West-Vlaanderen, Kortrijk, Belgium.

 

Résumé

L’examen de la goutte épaisse colorée est l’une des techniques les plus utilisées pour le diagnostic parasitologique de la trypanosomose africaine. Pourtant la technique est laborieuse et la reconaissance du trypanosome peut-être difficile à cause des artéfacts et des déformations morphologiques du parasite. Cependant, nous avons mis au point un test d’immunofluorescence directe pour une lecture plus facile, plus rapide et plus fiable des gouttes épaisses.

 

Un boeuf à été infecté avec Trypanosoma brucei (T.b.) brucei AnTat 1.1A, traité au bérénil après trois semaines et immunisé avec une préparation brute de T.b. gambiense LiTat 1,5 et T. b. gambiense LiTat 1,3 après 4 et 7 semaines. Après dix semaines, le sérum était très réactif en immunofluorescence indirecte avec T. evansi, T.b. rhodesiense, T. congolense and T. vivax. Les anticorps sériques ont été purifiés par précipitation avec le sulfate d’ammonium. La fraction purifiée a été marquée fluorescente avec FITC, et la réactivité du conjugué à été de nouveau testée par immunofluorescence directe sur différentes espèces de trypanosomes. Le conjugué réagissait principalement avec les sous-espèces de T. brucei.

 

Des gouttes épaisses de 1 µl contenant 1, 4, 16 et 64 trypanosomes/µl ont été préparées. Certaines gouttes épaisses ont été colorées au Giemsa, d’autres ont été préparées par la technique DIFAT. Les préparations ont été lues par trois personnes. Le temps nécessaire pour la détection du premier trypanosome a été noté. Les lames ont été notées négatives si aucun trypanosome n’a été observé pendant dix minutes. Pour les gouttes épaisses contenant 4, 16 et 64 trypanosomes/µl, la méthode DIFAT est significativement plus rapide (respectivement 2.4, 9 et 35 fois) que la coloration de Giemsa. Avec la technique DIFAT seul le trypanosome est coloré dans la goutte épaisse, il en résulte une meilleure identification. Ceci permet un dépistage plus rapide et conduit à une meilleure sensibilité. Par sa réactivité avec les sous-espèces de T. brucei, DIFAT peut améliorer de manière significative le diagnostic de la trypanosomose africaine humaine. La disponibilité d'appareils à faible coût pour adapter des microscopes ordinaires à la fluorescence pourra faciliter l'application du DIFAT sur le terrain. L'amélioration de la technique et la stabilité du conjugué restent nécessaires.

 

Summary

Examination of a stained thick blood film is one of the most applied parasitological techniques for diagnosis of African trypanosomosis.  The technique is however laborious and trypanosome recognition may be difficult due to artifacts and morphological distortion of the parasite.  We, therefore, developed a direct immunofluorescence test for easier, more reliable and rapid reading of the thick blood film. 

 

A bovine was infected with Trypanosoma brucei (T.b.) brucei AnTat 1.1A, treated with berenil after 3 weeks and immunised with crude extracts of T.b. gambiense LiTat 1.5 and T.b. gambiense LiTat 1.3 after 4 and 7 weeks.  After 10 weeks, the serum was highly reactive in indirect immunofluorescence with T. evansi, T.b. rhodesiense, T. congolense and T.vivax.  Antibodies were purified from the serum by ammoniumsulfate precipitation.  The purified fraction was fluorescently labelled with FITC, and the reactivity of the conjugate was again tested in direct immunofluorescence on different trypanosome species.  The FITC conjugate was mainly reactive with T. brucei subspecies. 

 

One microliter thick blood film preparations with 1, 4, 16, and 64 trypanosomes/µl (T.b. brucei) were prepared and Giemsa stained or developed with DIFAT.  The preparations were examined by 3 persons. Reading time needed to detect the first trypanosome was recorded.  Slides were scored negative when no trypanosomes were detected within 10 minutes.  For thick blood film preparations with 4, 16, and 64 trypanosomes/µl, DIFAT was significantly quicker (respectively about 2.4, 9 and 35 times) than Giemsa staining.  In DIFAT only the trypanosome is stained in the tick blood film preparation, resulting in better recognition.  This allows quicker screening of the preparation and leads to improved sensitivity.

Due to its reactivity with the brucei-subspecies, DIFAT can significantly improve the diagnosis of human African trypanosomosis.  The availability of simple, low-cost fluorescence devices to adapt to ordinary microscopes will also make it applicable under field conditions.  Further improvement, stabilisation and testing of the conjugate remain necessary.

 

Introduction

Definite diagnosis of African trypanosomosis still relies on detection of the parasite in body fluids.  Examination of thick blood film is one of the most applied parasitological techniques since it does not require special equipment such as a centrifuge, and there is no need for electrical power.  Examination of the stained slides is however laborious and trypanosome recognition may be difficult.  Apart from the trypanosomes, also white blood cells are stained and parasites may be difficult to recognize due to morphological distortion and artifacts.

 

Reading of the thick blood film would be facilitated if only trypanosomes appear stained.  This can be obtained by the use of labelled, specific antibodies, binding only to trypanosomes.

 

We therefore generated trypanosome specific antibodies in a bovine and labelled them with FITC in order to develop a direct immunofluorescence test (DIFAT).  Staining with DIFAT was compared to Giemsa on ease and speed of trypanosome detection in a thick blood film.

 

Materials and Methods

 

Generation of an immune serum

A bovine was infected intravenously with T.b. brucei AnTat 1.1A (100,000 trypanosomes) and was treated with Berenil after 3 weeks.  Four weeks post-infection a pellet of pure T.b. gambiense LiTat 1.5 was sonicated and was used to immunise the bovine intramuscularly.  The bovine was further immunised intravenously on week 7, with a non-sonicated pellet of 1 ml of pure T.b. gambiense LiTat 1.3.  10 weeks post-infection, 0.5 l of blood was taken to prepare the immune serum used for experiments described below.

 

Thin blood films

Thin blood films were prepared with blood from mice experimentally infected with T. evansi AnTat 3.1, T. evansi RoTat 1.2, T.b. rhodesiense ETat1.2R, T.b. gambiense LiTaT 1.3, T. congolense Lion 209 and T.vivax ILRAD 700.  Slides were air dried, fixed in aceton, wrapped in hygienic paper, sealed in a plastic bag with dessicant and stored at –20°C until use.

 

Indirect immunofluorescence

Thin blood films were thawn for at least 15 min and reaction zones of 0.5 cm x 0.5 cm were marked out using nail varnish.  After drying for 30 minutes, slides were rehydrated for 15 minutes in PBS (pH 7.2).  The buffer was removed between the reaction zones, and 30 µl of 1:100 serum dilution in PBS was incubated for 30 minutes.  Slides were rinsed 2 times in PBS and washed 2 times for 5 minutes in PBS.  Buffer was removed between the reaction zones before adding 30 µl/zone of anti-bovine IgG (H+L)-FITC diluted 1:50 in PBS containing 0.01% w/v Evans Blue.  After 30 minutes of incubation, slides were rinsed twice and washed twice as before.  The preparation was covered with a drop of buffered glycerol (1 volume of glycerol 87% + 1 volume of PBS) and a coverslip.  The immunofluorescence was scored under a fluorescence microscope at magnification 10 x  50 using a water objective.

 

Ammonium sulphate precipitation

The serum was centrifuged for 30 min at 3,000 g.  Ammonium sulphate was added to the supernatant to a final concentration of 40% and the solution was mixed overnight at 4°C.  The solution was centrifuged for 30 min at 3,000 g and the pellet containing the immunoglobulin was resuspended in water.  The remaining ammonium sulphate was removed during a buffer exchange with physiological salt solution on an Amicon ultrafiltration cell (PM30 membrane), followed by dialysis.  The remaining sample was centrifuged at 6,160 g for 30 minutes and the protein concentration (1.55 x OD280 - 0.76 x OD260) in the supernatant was measured in a spectrophotometer at 280 and 260 nm.

 

FITC coupling

The purified IgG was diluted to a concentration of 2 mg/ml in carbonate buffer (0.1M, pH 9).  Per mg of IgG, 50 µl of a 0.5% FITC solution in DMSO was added.  After coupling for 2 h, the IgG-FITC conjugate was separated from unbound FITC using a sephadex column (Pharmacia).  The column was eluated with PBS, and fractions of 0.5 ml were collected.  The protein concentration ((OD280 - OD495 x 0.35) / 1.35) and FITC/IgG ratio (OD495 / OD280) of the eluated fractions were determined in a spectrophotometer at 495 and 280 nm.  Fractions with high protein and FITC concentration were pooled.  For storage, 50% glycerol was added to a final concentration of 5%.  The solution was filtered over a 0.22 µm filter to remove particles, and stored at 4°C until use.

 

Direct immunofluorescence (DIFAT)

For direct immunofluorescence, IgG-FITC was diluted in PBS containing 0.01% w/v Evans Blue and applied on the thin or thick blood films after rehydratation (see protocol indirect immunofluorescence).  The slides were mounted as described above for microscopic examination.

 

DIFAT versus Giemsa staining of thick blood films

One microliter thick blood films were prepared with mouse blood containing 1, 4, 16, and 64 trypanosomes/µl (T.b. brucei).  For DIFAT, thick blood films were fixed in aceton and frozen; for Giemsa staining slides were frozen without fixation.

 

For comparison of DIFAT versus Giemsa, slides were thawn and Giemsa stained or developed with DIFAT (1:2 dilution).  The preparations were examined by 3 persons (2 experienced, 1 unexperienced).  15 preparations of each trypanosome dilution were examined per person with Giemsa and DIFAT.  Reading time needed to detect the first trypanosome was recorded.  Slides were scored negative when no trypanosomes were detected within 10 minutes.

 

Results

 

Initial testing of immune serum in indirect fluorescence

The bovine immunization serum was checked for reactivity with trypanosomes by indirect immunofluorescence.  The serum reacted strongly with all trypanosome species and subspecies tested (Table 1).  The complete trypanosome cells appeared fluorescent and nucleus, kinetoplast and/or the flagellum were clearly visible.  No background fluorescence was observed.  Based on this result, the immunoglobulins in the immune serum were purified by ammonium sulphate precipitation and dialysis.  A typical yield of 1300 mg immunoglobulin was obtained from 20 ml serum.

 

Table 1: Intensity of indirect immunofluorescence with the bovine immune serum and different trypanosomes species.

 

Trypanosome species

Immune serum 1:100

Week 10

Pre-immune serum 1:100

Week 0

T. evansi AnTat 3.1

+++

+/-

T. evansi RoTat 1.2

+++

+/-

T.b. rhodesiense ETat 1.2R

+++

+/-

T. congolense Lion 209

+++

+/-

T. vivax ILRAD 700

+++

+/-

 

FITC coupling

The obtained optical densities (O.D) after FITC coupling and sephadex column purification are shown in Table 2.  Fractions 1-5 contained sufficient protein and FITC and were pooled, resulting in a conjugate with 0.763 mg/ml protein concentration. 

 

Table 2: O.D. at 280 and 495 nm, FITC/IgG ratio and protein concentration of different IgG-FITC fractions purified over a sephadex column.

 

Fraction

O.D. 280 nm

O.D. 495 nm

FITC/IgG

IgG (mg/ml)

1

1.558

2.238

1.436

0.574

2

2.469

>2.5

N.D.*

N.D.*

3

> 2.5

>2.5

N.D.*

N.D.*

4

2.264

2.487

1.098

1.032

5

0.585

0.970

1.658

0.182

6

0.064

0.106

1.656

0.020

7

0.043

0.076

1.767

0.012

*Not determined since O.D above maximum

 

The FITC-IgG conjugate was then tested again in different dilutions on different trypanosome species (Table 3).  The IgG-FITC conjugate reacted strongly with T. evansi and T.b. rhodesiense in 1:5 - 1:20 dilutions.  Reactions with T.b. gambiense and T. congolense were positive at 1:5 and 1:10.  The reaction with T. vivax was weak while some background fluorescence was observed.

 

Table 3: Immunofluorescence intensity observed in DIFAT on different trypanosomes species using different conjugate dilutions.

 

Trypanosome species

Dilution 1:5

Dilution 1:10

Dilution 1:20

Dilution 1:40

T. evansi AnTat 3.1

++

++

+

-

T. evansi RoTat 1.2

++

++

+

+/-

T.b.r ETat 1.2R

++

++

+

+/-

T.b.g. LiTat 1.3

+

+

+/-

-

T. congolense lion 209

+

+

+/-

-

T. vivaxi ILRAD 700

+/-

+/-

-

-

 

Comparative study of direct immunofluorescence versus Giemsa

In thick blood films containing 64, 16, 4, and 1 trypanosomes/µl, the median time to detect the first trypanosome with Giemsa was respectively 35, 105, 200 and 600 seconds. With DIFAT, median time was 1, 12, 85 and 280 seconds (Figure 1).  In preparations with 64, 16, and 4 trypanosomes/µl, the parasites were detected significantly faster with DIFAT than with Giemsa (respectively 35, 9 and 2.4 times). 

When thick drop films were stained with DIFAT, trypanosomes could be found back in all thick blood films containing 64, 16 or 4 trypanosomes/µl.  In preparations containing 1 trypanosome/µl, trypanosomes were found back within 600 seconds in 33/45 cases.  With Giemsa staining, trypanosomes were found only in 43/45, 36/45 and 13/45 thick blood films containing respectively 16, 4 or 1 trypanosomes/µl.

 

Figure 1: Median reading times for tick drop preparations containing different trypanosome concentrations, stained with DIFAT (white boxes) or Giemsa (grey boxes).  The proportion of positive results with each technique is indicated next to the bars.