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Immunofluorescence and counter immunoelectrophoresis in the diagnosis of kala-azar
149 TRANSAC?IONS
OF THE ROYAL
SOCIETY
Immunofluorescence HAMID
Departme~~ts
R.
REZAI,
oJ’ Microbiology
OF
TROPICAL MEDICINE AND HYGIENE, VOL. 71, NO. 2,1977.
and counter immunoelectrophoresis the diagnosis of kala-azar
in
N. BEHFOROUZ,G. H. AMIRHAKIMI AND J. KOHANTEB and Pediatricx
Summary Techniques of indirect immunofluorescence (IF) and counter current immunoelectrophoresis (CIEP) were employed and compared for the diagnosis of kala-azar among children in Fars Province of Iran. It is suggested that the diagnostic titre for kala-azar by immunofluorescence is over 1 in 64. In fact a majority of the cases had a titre of more than 1 in 256. ClEP using soluble antigens prepared from the promastigote form of Lei.shmunia donovani, was also applied to sera from patients with systemic leishmaniasis and other diseases. The CIEP test correlated well with the indirect immunofluorescent test. It is therefore suggested that CIEP, which is a rapid and less sophisticated test, can be applied for the diagnosis of kala-azar cases and epidemiological surveys. Introduction With the cessation of the anti-malaria programme in Iran, the incidence of systemic leishmaniasis among children in Fars Province of Iran has risen. Whereas a few years ago, there were usually no more than 12 cases per year, we are now beginning to see up to three cases a month in the Pahlavi University Hospitals in Shiraz. Unfortunately, diagnosis of visceral leishmaniasis is often difficult both clinically and in the laboratory. Many of the clinical features of kala-azar are common to other diseases and the parasites are not easily demonstrated from clinical specimens. Many of the serological tests used in diagnosis measure the non-specific rise in serum globulins which are often associated with visceral leishmaniasis. Globulin precipitation, flocculation and the aldehyde test are examples of such techniques and have only limited diagnostic value (HENRY, 1953; NAPIER,1922). The indirect fluorescent antibody (IFA) test has been reported to be the superior test for the detection of cutaneous, mucocutaneous and systemic leishmaniasis (BRAY & LAINSON, 1965; SHAW & VOLLER, 1964; WALTON et al., 1972), although some cross reactions apparently do occur (DUXBURY& SADUN, 1964). Counter current immunoelectrophoresis (CIEP), a rapid and unsophisticated technique, has been used for the detection of Australia antigen (GOCKE & CALDERON,1970) and more recently has been applied to the diagnosis of some parasitic infections (DESOWITZet al., 1975). In the present report, both tests were applied to the diagnosis of patients suspected of having kala-azar. The diagnostic value of the IFA technique was confirmed and the range of significant titres for this test was determined. CIEP was also applied to the detection of systemic leishmaniasis in these patients and the results were compared to those of the IFA test.
Medical
School,
Pahlakxi University,
Shiraz,
Iran
Materials and methods Sera
Over a period of 18 months, a total of 40 sera were collected from children suspected of having visceral leishmaniasis. Sera were also collected from children with a variety of other infections or diseases particularly those having hepatosplenomegaly or blood disorders. Techniques of immunofluorescence
The IFA technique was used on all sera as described in our previous report (BEHFOROUZ e/ al., 1976). Conjugated antisera was obtained from the Nordic Laboratories. Preparation of soluble fintigenjor counter current immunoelectrophoresis Lei~shmanin donovani was grown in Panmede media & WHITTINGTON, 1957). After four to five days, the Leishmania were centrifuged, washed and the number was adjusted to 16 x 107per cc. The Leishmania were disrupted by ultrasonication and freezing and thawing. The suspension was centrifuged, the supernatant was dialysed against phosphate buffered saline (PBS) and concentrated to one-third of the original volume. The gel plates were prepared from 0.85% agarose dissolved in barbital-acetate buffer pH 8.2, M-0.05. The electrophoresis was performed in an Austigen (Hyland) apparatus using 0.1 M barbital-acetate buffer, pH 8.2. The wells were spaced 4.0 mm apart so that IO samples could be tested on each plate. Before testing, all serum samples were heat inactivated at 56°C for 30 minutes. The serum samples were placed in the anodic wells and the soluble antigen in the cathodic wells. Electrophoresis was carried out at 40mA for 30 minutes and the plates were immediately examined for precipitin bands. The plates were then stored at 4’ C and re-examined after 24 hours.
(FEINBERG
Results Puraclitricaily
proven cases of kab-mar
The sera from 12 patients who had clincial symptoms of kala-azar including fever, hepatosplenomegaly, low haemoglobin, reversed A/G ratio, and pancytopenia and who were shown either by bone marrow smears or culture to contain Leishmania, were studied by the IFA and CIEP techniques. 11 of these 12 patients were under eight years of age. Ail the sera of these patients had an immunofluorescent titre of l/64 or over; nine of 12 sera had a titre of l/256 or more. Although sera samples of only some of these patients were available for counter current immunoelectrophoresis, six of the seven sera tejted gave precipitin lines with the prepared antigen. Clinically
diagnosed cases of kala-azar
The sera of 23 children who, on admission were suspected of having kala-azar and had many of the same clinical symptoms as the first group but in whom Lei.sh-
150
IF AND CIEP IN DIAGNOSIS
Table I - Immunofluorescence kala-azar IFA
titre
No. of cases
OF KALA-AZAR
and counter current immunoelectrophoresis
Final clinical diagnosis
on the sera of cases clinically
Glucantime treatment
CIEP test Pos. *
Neg. **
Undiagnosed lymphoadenopathy (possibly kala-azar)
0
1
2
0
Not tested _____-
diagnosed to be
Other medication --__Not Resp. resp. *** ***
Resp. ***
Not resp. ***
0
I
-
3
0
0
lin 64
1
1 in 128
4
All kala-azar
lin 256
11
10 kala-azar One died before final diagnosis (probably kala-azar)
9
0
2
I
0
0
All kala-azar
5
0
2
4
0
1 given flaw1
1 in 512 Total cases
0 ---__ 2
____
7
23
*Positive
16 **Negative
1
___________-
1
6
14
1
1
2 -__1 -__4
***Responded
were not detected, were studied serologically by the IFA technique and in some cases, by CIEP. Almost all of these patients were children under eight years of age. Table I presents the serological findings, the final clinical diagnosis, treatment and outcome of these patients. 22 of these patients’ sera showed a titre of l/128 or above by the IFA test. 14 of these patients were given glucantime and six were either not treated or were given sntiblcterial drugs (the treatment of two of these cases is unknown). Glucantime is a pentavalent antimony compound which is the drug of choice for leishmanial infections. All those treated with glucantime survived except one. Four of five of the non-glucantime treated. IFA-positive patients did not respond to alternate treatment and died. Although the CIEP test was not performed on the sera of all these patients, all sera with a titre of 128 or above which were tested were found to give precipitin lines with the prepared L. donovani antigen. The one patient with an IFA titre of l/64 was treated with glucantime but died with no final diagnosis. The CIEP test on this child was negative. Based on their positive response to glucantime, their typical kala-azar symptoms and their positive IFA tests, it can probably be assumed that these patients were suffering from systemic leishmaniasis, in spite of the negative biopsy reports. The CIEP test correlated well with the results of the IFA test and was helpful in confirming our laboratory diagnosis. mania
Control groups
In this group, sera from children with a variety of other diseases as well as sera from five normal individuals
were examined by the two immunological tests and the results indicated that the titre of IFA was not over l/32 in 27 sera tested. 23 of these sera had a titre of l/16 or below, with low intensities of fluorescence and CIEP test was negative. The techniques of immunofluorescence and counter current immunoelectrophoresis were also applied to the sera of guinea-pigs infected with Leishmania enriettii and mice infected with L. tropica. A guinea-pig in an advanced stage of infection with several metastatic lesions had an IFA titre of 1 in 512 and gave a heavy precipitin line by CIEP against the L. donovani antigen. Guinea-pigs in an early stage of infection, having IFA titre of 1 in 64, gave no precipitin line using CIEP. The sera from infected mice with an IFA titre of 1 in 256 were also unreactive using CIEP. Discussion
In the past few years, there have been an increasing number of cases of systemic leishmaniasis among children in Fars Province of Iran. Diagnosis of these cases is usually based on clinical symptoms such as fever, hepatosplenomegaly reversed A/G ratio, pancytopenia, as well as examination of bone marrow or spleen biopsy for Leishmania. The clinical diagnosis of kala-azar is often difficult because some other diseasesaffecting children may mimic several or all of the typical symptoms and, moreover, in some cases of true kala-azar some of the diagnostic symptoms may be absent. Direct examination of bone marrow for the presence of Leishmania is rarely successful and requires great expertise. Moreover proper aspiration of the bone marrow is painful for the child and
HAMID
R. REZAI
difficult for the physician. Although perhaps helpful, spleen biopsy may be dangerous and therefore not routinely done. Culture of clinical specimens is not usually successful, probably because of the presence of normal anti-leishmania factors in the blood used in culture media (REZAI et al., 1975). Therefore, there has been a great need for a more reliable laboratory test for diagnosis of systemic leishmaniasis in this area. The present study reports the application of the immunofluorescent technique and the development of a new test, counter current immunoelectrophoresis to the diagnosis of kala-azar among children. Patients were divided into three groups; those who were paraclinically proven to have kala-azar, those who were clinically diagnosed as having systemic leishmaniasis and the ones who were suffering from other disorders. In the first group, in which Leishmania had been detected either by direct examination or culture, all had a IFA titre of over I /64 and over 80 % had titres of l/256 or above. In the second group, over 90% of the children had a IFA titre of 128 or above. In the third group, who were shown to have other diseases, the IFA titre did not exceed l/32. On the basis of this data therefore, one can conclude that a titre of l/l28 or above could be used as a diagnostic level in systemic leishmaniasis. In all these three groups, the results of the counter current immunoelectrophoresis test correlated well with the immunofluorescent technique. We suggest that CIEP, a rapid and less sophisticated test than IFA, may be used for the laboratory diagnosis of systemic leishmaniasis and may possibly be applied to the epidemiological screening of large populations in less equipped laboratories. References Behforouz, N., Rezai, H. R. & Gettner, S. (1976). Application of immunofluorescence to detection of antibody in Leishmania infection. Annals of Tropical Medicine and Parasitology,
70,293-301.
I51
et a/.
Bray, R. S. & Lainson, R. (1965). The immunology and serology of leishmaniasis. The fluorescent antibody staining technique. Transactions of the Royal Society of Tropical Medicine and Hygiene, 59,535-544.
Desowitz, R. S., Draper, C. C. & Phillips, T. M. (1975). Preliminary observations on counter current immunoelectrophoresis of Trypanosoma cruzi and Leishmania. Transactions of the Roval Societv.” of TroDical Medicine . and Hygiene, 69,430. .
Duxbury, R. W. & Sadun, E. H. (1964). Fluorescent antibody test for the serodiagnosis of visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene, 13,525-529.
Feinberg, J. G. & Whittington, M. J. (1957). A culture medium for Trichomonas vagina/is Donnt and species of Candida. Journal of Clinical Pathology, 10, 327. Gocke, D. J. & Calderon, H. (1970). Rapid detection of Australia antigen by counterimmunoelectrophoresis. Journal of Immunology, 104B, 103I-1034. Henery, A. F. X. (1953). Kala-azar et paludofloculation. Revue du Paludisme et de MPdecine 11,7-9.
Tropicale,
Paris,
Napier, L. E. (1922). A new serum test for kala-azar. Indian Journal of Medical
Research, 9, 830.
Prekarski, G., Saathoff, M. & Nouri Nekoui, M. H. (1973). Studies on the detection of antibodies in kala-azar patients and animals experimentally infected with Leishmania donovani. Tropenmedizin und Parasitologie, 24, 161~173. Rezai, H. R., Ardehali, S. & Gettner, S. (1975). Antileishmania activity of normal animal sera. Annals of TropicalMedicine
and Parasitology,
69,29-33.
Shaw, J. J. & Voller, A. (1964). The detection of circulating antibody to kala-azar by means of immunofluorescent techniques. Transactions of the Royal Society of Tropical Medicine and Hygiene, 58, 349-352. Arreptedfbr
publication
28th October, 1976.
OF THE ROYAL
SOCIETY
Immunofluorescence HAMID
Departme~~ts
R.
REZAI,
oJ’ Microbiology
OF
TROPICAL MEDICINE AND HYGIENE, VOL. 71, NO. 2,1977.
and counter immunoelectrophoresis the diagnosis of kala-azar
in
N. BEHFOROUZ,G. H. AMIRHAKIMI AND J. KOHANTEB and Pediatricx
Summary Techniques of indirect immunofluorescence (IF) and counter current immunoelectrophoresis (CIEP) were employed and compared for the diagnosis of kala-azar among children in Fars Province of Iran. It is suggested that the diagnostic titre for kala-azar by immunofluorescence is over 1 in 64. In fact a majority of the cases had a titre of more than 1 in 256. ClEP using soluble antigens prepared from the promastigote form of Lei.shmunia donovani, was also applied to sera from patients with systemic leishmaniasis and other diseases. The CIEP test correlated well with the indirect immunofluorescent test. It is therefore suggested that CIEP, which is a rapid and less sophisticated test, can be applied for the diagnosis of kala-azar cases and epidemiological surveys. Introduction With the cessation of the anti-malaria programme in Iran, the incidence of systemic leishmaniasis among children in Fars Province of Iran has risen. Whereas a few years ago, there were usually no more than 12 cases per year, we are now beginning to see up to three cases a month in the Pahlavi University Hospitals in Shiraz. Unfortunately, diagnosis of visceral leishmaniasis is often difficult both clinically and in the laboratory. Many of the clinical features of kala-azar are common to other diseases and the parasites are not easily demonstrated from clinical specimens. Many of the serological tests used in diagnosis measure the non-specific rise in serum globulins which are often associated with visceral leishmaniasis. Globulin precipitation, flocculation and the aldehyde test are examples of such techniques and have only limited diagnostic value (HENRY, 1953; NAPIER,1922). The indirect fluorescent antibody (IFA) test has been reported to be the superior test for the detection of cutaneous, mucocutaneous and systemic leishmaniasis (BRAY & LAINSON, 1965; SHAW & VOLLER, 1964; WALTON et al., 1972), although some cross reactions apparently do occur (DUXBURY& SADUN, 1964). Counter current immunoelectrophoresis (CIEP), a rapid and unsophisticated technique, has been used for the detection of Australia antigen (GOCKE & CALDERON,1970) and more recently has been applied to the diagnosis of some parasitic infections (DESOWITZet al., 1975). In the present report, both tests were applied to the diagnosis of patients suspected of having kala-azar. The diagnostic value of the IFA technique was confirmed and the range of significant titres for this test was determined. CIEP was also applied to the detection of systemic leishmaniasis in these patients and the results were compared to those of the IFA test.
Medical
School,
Pahlakxi University,
Shiraz,
Iran
Materials and methods Sera
Over a period of 18 months, a total of 40 sera were collected from children suspected of having visceral leishmaniasis. Sera were also collected from children with a variety of other infections or diseases particularly those having hepatosplenomegaly or blood disorders. Techniques of immunofluorescence
The IFA technique was used on all sera as described in our previous report (BEHFOROUZ e/ al., 1976). Conjugated antisera was obtained from the Nordic Laboratories. Preparation of soluble fintigenjor counter current immunoelectrophoresis Lei~shmanin donovani was grown in Panmede media & WHITTINGTON, 1957). After four to five days, the Leishmania were centrifuged, washed and the number was adjusted to 16 x 107per cc. The Leishmania were disrupted by ultrasonication and freezing and thawing. The suspension was centrifuged, the supernatant was dialysed against phosphate buffered saline (PBS) and concentrated to one-third of the original volume. The gel plates were prepared from 0.85% agarose dissolved in barbital-acetate buffer pH 8.2, M-0.05. The electrophoresis was performed in an Austigen (Hyland) apparatus using 0.1 M barbital-acetate buffer, pH 8.2. The wells were spaced 4.0 mm apart so that IO samples could be tested on each plate. Before testing, all serum samples were heat inactivated at 56°C for 30 minutes. The serum samples were placed in the anodic wells and the soluble antigen in the cathodic wells. Electrophoresis was carried out at 40mA for 30 minutes and the plates were immediately examined for precipitin bands. The plates were then stored at 4’ C and re-examined after 24 hours.
(FEINBERG
Results Puraclitricaily
proven cases of kab-mar
The sera from 12 patients who had clincial symptoms of kala-azar including fever, hepatosplenomegaly, low haemoglobin, reversed A/G ratio, and pancytopenia and who were shown either by bone marrow smears or culture to contain Leishmania, were studied by the IFA and CIEP techniques. 11 of these 12 patients were under eight years of age. Ail the sera of these patients had an immunofluorescent titre of l/64 or over; nine of 12 sera had a titre of l/256 or more. Although sera samples of only some of these patients were available for counter current immunoelectrophoresis, six of the seven sera tejted gave precipitin lines with the prepared antigen. Clinically
diagnosed cases of kala-azar
The sera of 23 children who, on admission were suspected of having kala-azar and had many of the same clinical symptoms as the first group but in whom Lei.sh-
150
IF AND CIEP IN DIAGNOSIS
Table I - Immunofluorescence kala-azar IFA
titre
No. of cases
OF KALA-AZAR
and counter current immunoelectrophoresis
Final clinical diagnosis
on the sera of cases clinically
Glucantime treatment
CIEP test Pos. *
Neg. **
Undiagnosed lymphoadenopathy (possibly kala-azar)
0
1
2
0
Not tested _____-
diagnosed to be
Other medication --__Not Resp. resp. *** ***
Resp. ***
Not resp. ***
0
I
-
3
0
0
lin 64
1
1 in 128
4
All kala-azar
lin 256
11
10 kala-azar One died before final diagnosis (probably kala-azar)
9
0
2
I
0
0
All kala-azar
5
0
2
4
0
1 given flaw1
1 in 512 Total cases
0 ---__ 2
____
7
23
*Positive
16 **Negative
1
___________-
1
6
14
1
1
2 -__1 -__4
***Responded
were not detected, were studied serologically by the IFA technique and in some cases, by CIEP. Almost all of these patients were children under eight years of age. Table I presents the serological findings, the final clinical diagnosis, treatment and outcome of these patients. 22 of these patients’ sera showed a titre of l/128 or above by the IFA test. 14 of these patients were given glucantime and six were either not treated or were given sntiblcterial drugs (the treatment of two of these cases is unknown). Glucantime is a pentavalent antimony compound which is the drug of choice for leishmanial infections. All those treated with glucantime survived except one. Four of five of the non-glucantime treated. IFA-positive patients did not respond to alternate treatment and died. Although the CIEP test was not performed on the sera of all these patients, all sera with a titre of 128 or above which were tested were found to give precipitin lines with the prepared L. donovani antigen. The one patient with an IFA titre of l/64 was treated with glucantime but died with no final diagnosis. The CIEP test on this child was negative. Based on their positive response to glucantime, their typical kala-azar symptoms and their positive IFA tests, it can probably be assumed that these patients were suffering from systemic leishmaniasis, in spite of the negative biopsy reports. The CIEP test correlated well with the results of the IFA test and was helpful in confirming our laboratory diagnosis. mania
Control groups
In this group, sera from children with a variety of other diseases as well as sera from five normal individuals
were examined by the two immunological tests and the results indicated that the titre of IFA was not over l/32 in 27 sera tested. 23 of these sera had a titre of l/16 or below, with low intensities of fluorescence and CIEP test was negative. The techniques of immunofluorescence and counter current immunoelectrophoresis were also applied to the sera of guinea-pigs infected with Leishmania enriettii and mice infected with L. tropica. A guinea-pig in an advanced stage of infection with several metastatic lesions had an IFA titre of 1 in 512 and gave a heavy precipitin line by CIEP against the L. donovani antigen. Guinea-pigs in an early stage of infection, having IFA titre of 1 in 64, gave no precipitin line using CIEP. The sera from infected mice with an IFA titre of 1 in 256 were also unreactive using CIEP. Discussion
In the past few years, there have been an increasing number of cases of systemic leishmaniasis among children in Fars Province of Iran. Diagnosis of these cases is usually based on clinical symptoms such as fever, hepatosplenomegaly reversed A/G ratio, pancytopenia, as well as examination of bone marrow or spleen biopsy for Leishmania. The clinical diagnosis of kala-azar is often difficult because some other diseasesaffecting children may mimic several or all of the typical symptoms and, moreover, in some cases of true kala-azar some of the diagnostic symptoms may be absent. Direct examination of bone marrow for the presence of Leishmania is rarely successful and requires great expertise. Moreover proper aspiration of the bone marrow is painful for the child and
HAMID
R. REZAI
difficult for the physician. Although perhaps helpful, spleen biopsy may be dangerous and therefore not routinely done. Culture of clinical specimens is not usually successful, probably because of the presence of normal anti-leishmania factors in the blood used in culture media (REZAI et al., 1975). Therefore, there has been a great need for a more reliable laboratory test for diagnosis of systemic leishmaniasis in this area. The present study reports the application of the immunofluorescent technique and the development of a new test, counter current immunoelectrophoresis to the diagnosis of kala-azar among children. Patients were divided into three groups; those who were paraclinically proven to have kala-azar, those who were clinically diagnosed as having systemic leishmaniasis and the ones who were suffering from other disorders. In the first group, in which Leishmania had been detected either by direct examination or culture, all had a IFA titre of over I /64 and over 80 % had titres of l/256 or above. In the second group, over 90% of the children had a IFA titre of 128 or above. In the third group, who were shown to have other diseases, the IFA titre did not exceed l/32. On the basis of this data therefore, one can conclude that a titre of l/l28 or above could be used as a diagnostic level in systemic leishmaniasis. In all these three groups, the results of the counter current immunoelectrophoresis test correlated well with the immunofluorescent technique. We suggest that CIEP, a rapid and less sophisticated test than IFA, may be used for the laboratory diagnosis of systemic leishmaniasis and may possibly be applied to the epidemiological screening of large populations in less equipped laboratories. References Behforouz, N., Rezai, H. R. & Gettner, S. (1976). Application of immunofluorescence to detection of antibody in Leishmania infection. Annals of Tropical Medicine and Parasitology,
70,293-301.
I51
et a/.
Bray, R. S. & Lainson, R. (1965). The immunology and serology of leishmaniasis. The fluorescent antibody staining technique. Transactions of the Royal Society of Tropical Medicine and Hygiene, 59,535-544.
Desowitz, R. S., Draper, C. C. & Phillips, T. M. (1975). Preliminary observations on counter current immunoelectrophoresis of Trypanosoma cruzi and Leishmania. Transactions of the Roval Societv.” of TroDical Medicine . and Hygiene, 69,430. .
Duxbury, R. W. & Sadun, E. H. (1964). Fluorescent antibody test for the serodiagnosis of visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene, 13,525-529.
Feinberg, J. G. & Whittington, M. J. (1957). A culture medium for Trichomonas vagina/is Donnt and species of Candida. Journal of Clinical Pathology, 10, 327. Gocke, D. J. & Calderon, H. (1970). Rapid detection of Australia antigen by counterimmunoelectrophoresis. Journal of Immunology, 104B, 103I-1034. Henery, A. F. X. (1953). Kala-azar et paludofloculation. Revue du Paludisme et de MPdecine 11,7-9.
Tropicale,
Paris,
Napier, L. E. (1922). A new serum test for kala-azar. Indian Journal of Medical
Research, 9, 830.
Prekarski, G., Saathoff, M. & Nouri Nekoui, M. H. (1973). Studies on the detection of antibodies in kala-azar patients and animals experimentally infected with Leishmania donovani. Tropenmedizin und Parasitologie, 24, 161~173. Rezai, H. R., Ardehali, S. & Gettner, S. (1975). Antileishmania activity of normal animal sera. Annals of TropicalMedicine
and Parasitology,
69,29-33.
Shaw, J. J. & Voller, A. (1964). The detection of circulating antibody to kala-azar by means of immunofluorescent techniques. Transactions of the Royal Society of Tropical Medicine and Hygiene, 58, 349-352. Arreptedfbr
publication
28th October, 1976.