Sero-epidemiological assessment and diagnosis of visceral leishmaniasis in an endemic locality using Fast Agglutination Screening Test (FAST)

Acta Tropica 83 (2002) 93 – 101 www.parasitology-online.com

Sero-epidemiological assessment and diagnosis of visceral leishmaniasis in an endemic locality using Fast Agglutination Screening Test (FAST) A. Hailu a,c,*, C.C.M. Kroon b, G.J. Schoone b, N. Berhe a, H.D.F.H. Schallig b, P.A. Kager c a

Leishmaniasis Research Group, Institute of Pathobiology, Addis Ababa Uni6ersity, P.O. Box 1176, Addis Ababa, Ethiopia b KIT (Koninklijk Instituut 6oor de Tropen/Royal Tropical Institute) Biomedical Research, Amsterdam, The Netherlands c Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Uni6ersity of Amsterdam, Amsterdam, The Netherlands Received 10 August 2001; received in revised form 1 February 2002; accepted 12 February 2002

Abstract The Fast Agglutination Screening Test (FAST) was employed on sera obtained from an endemic area of visceral leishmaniasis in southwestern Ethiopia, in February 2000. The study involved (i) active case detection among 1575 residents of two villages; and (ii) passive case detection in an outpatient clinic. Sera of 1587 individuals, including 143 sera of previously treated VL patients, were tested. Based on the size of agglutination mat, the FAST results were read qualitatively as non-reactive ( − ), weakly reactive (1 + ), moderately reactive (2 + ) and highly reactive (3 + ). All FAST reactive sera were re-tested with the Direct Agglutination Test (DAT). After clinical screening of 1625 individuals, 61 individuals with signs and symptoms of early or late VL were found; 26 sera were FAST positive. Twenty-two of these suspected VL cases were subjected to parasitological examination using lymph node aspirates. Eighteen (81.8%) were confirmed either by demonstration of amastigotes in smears or promastigotes in NNN cultures. FAST reactive anti-leishmanial antibodies were detected in 4.5% of untreated and 70.6% of previously treated patients. Forty-five sera of 1390 previously untreated asymptomatic individuals (3.2%) were found to be FAST positive. This report demonstrates that FAST is a rapid and cost-effective screening test for the diagnosis and sero-epidemiological surveillance of visceral leishmaniasis. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Direct Agglutination Test; Ethiopia; Fast Agglutination Screening Test; Visceral leishmaniasis; Sero-epidemiology

1. Introduction

* Corresponding author. Tel.: +251-1-763-091; fax: + 2511-755-296. E-mail address: [email protected] (A. Hailu).

VL is a chronically debilitating disease characterized by splenomegaly, prolonged fever, anemia, pancytopenia and weight loss (Boelaert et al., 2000). Since untreated VL is invariably fatal and

0001-706X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 2 ) 0 0 0 6 3 - 3

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as the currently available treatment options are expensive, an accurate diagnosis of the disease is mandatory. Clinical diagnosis of the disease is concluded by the demonstration of Leishmania parasites in the culture or smear of splenic, lymph node or bone marrow aspirates. These parasitological methods are undoubtedly very specific, but sensitivity is variable and can be as low as 50– 60% (Siddig et al., 1988, 1990; Zijlstra et al., 1992) depending on the type of tissue and methods employed. The aspiration procedures are invasive; in particular, splenic aspiration needs to be carried out by experienced clinicians, as it can be a risky procedure. The development of serological tests has helped to improve the diagnostic procedures. However, many of the currently available serological tests are not yet sufficiently simple and robust to be within the reach of health facilities. As a result, in many clinical situations, diagnosis of visceral leishmaniasis still depends on clinical criteria and a subsequent parasitological confirmation. When the tests are available, they are mostly used to confirm or rule out out clinical diagnosis. Over the last 15 years, the Direct Agglutination Test (DAT) has proved to be a very important sero-diagnostic tool, combining high levels of intrinsic validity and ease of performance (Harith et al., 1988; El Safi and Evans, 1989; Zijlstra et al., 1991; Boelaert et al., 1999). The test has so far been a semi-quantitative test, where test readings are given in end-point titres. The reading of end-point titres is bound to involve subjective errors, thus presenting a problem in the reproducibility of the test. Nevertheless, the test has been in field use in many endemic areas. We have recently described a modified version of DAT, the Fast Agglutination Screening Test (FAST), which we have shown to maintain the high levels of intrinsic validity described for DAT (Schoone et al., 2001). This test basically employs the DAT in a system designed to use a single serum dilution and in which test results are available within 3 h. The test was developed especially for application in epidemic situations and for screening of large populations. We report here the first set of data about its use in diagnosis and epidemiological screening of VL.

2. Materials and methods

2.1. Study design FAST was applied on sera obtained during a community-based study of VL in an endemic area in Ethiopia. The focus of VL in the Segen valley, south-western Ethiopia, has been under surveillance by the Leishmaniasis Research Group (LRG) of the Institute of Pathobiology (IPB) since 1982. This focus comprises a major part of the Konso District, in which a population of at least 70,000 people are considered at risk of infection by Leishmania dono6ani. The LRG has established a research and treatment centre in a village named Aba-Roba, where patients self-present to the centre four times a year. In addition, since August 1997, the group has been pursuing a community based study of natural L. dono6ani infections of humans in two high risk villages, Galga and Goinada, in which serological screening of the study population was carried out at 6 monthly intervals. This was combined with clinical screening and leishmanin skin test administered to all participants of the study. In February 2000, 1575 individuals (755 males, 820 females) reported in a cohort that comprised all residents of Galga and Goinada (Table 1). In addition, 50 other patients (40 males, 10 females) with complaints of a big spleen, fever and other constitutional symptoms, self-presented to the field centre. A total of 1587 sera were collected, which also included sera of 143 previously treated VL patients. Past history of treatment for all individuals of this community was available in the registry of the Institute of Pathobiology. All subjects with splenomegaly and fever of at least 2 weeks duration were considered as VL suspects. The sera of these subjects were tested in the field using DAT. Clinical suspects of VL with positive DAT were presumed as cases of VL (if previously untreated) and as probable cases of relapse or unresponsiveness (if treated before). Smear and culture of lymph node aspirates were obtained from all presumed cases of VL. Diagnosis was confirmed by demonstration of amastigotes in Giemsa stained smears of aspirates or promastigotes in NNN cultures maintained for 3

A. Hailu et al. / Acta Tropica 83 (2002) 93–101

weeks. For each lymph node aspirate, at least 5000 microscope fields were examined under oil immersion (magnification 1000× ). On average, this required 3 h of slide reading per patient. VL cases whose parasitological findings were positive were treated with a standard regimen of sodium stibogluconate, 20 mg/kg b.wt per day for 30 days. Cases with negative parasitological results were referred to a VL clinic of the district for further follow-up.

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agglutination wells were scored on the basis of three distinct sizes of agglutination mat: 3+ (large agglutination mat), 2+ (small agglutination mat) and 1+ (shrunken agglutination mat lacking haziness). DAT was performed as described previously (Harith et al., 1988; Meredith et al., 1995). In the field, sera of suspected VL cases were tested using liquid DAT antigen prepared at the Institute of Pathobiology in Addis Ababa (batch No. 23, 06/10/99). All FAST reactive sera were re-tested in the laboratory with conventional DAT using a freeze-dried antigen of KIT (lot of 04/10/99). As an internal control, a random sample of 30 sera with negative FAST results, which included ten sera of previously treated VL patients, were fully titrated using DAT. Titres were expressed in discrete numerical scales of small integers 0–10, from lowest of 1/100 to a high of 1/102,400. Cut-off titre was at 1/1600 (scale of 4) as locally adopted in Ethiopia (unpublished). Titres higher than 1/102,400 were assigned a scale of 11. Likewise, titres B 1/100 were assigned a scale of − 1.

2.2. FAST and DAT procedures All 1587 sera were tested by FAST using a freeze-dried antigen produced at the Koninklijk Instituut voor de Tropen/Royal Tropical Institute (KIT) Biomedical Research, Amsterdam, The Netherlands (KIT lot of 01/04/97). The test antigen contained formalin fixed Coomassie blue stained promastigotes of L. dono6ani (MHOM/ SD/68/1S). Details of antigen preparation are as described before (Schoone et al., 2001). Vials containing 2×108 promastigotes of the antigen were reconstituted in 1.0 ml normal saline to which 10 ml fetal calf serum was added. Using v-shaped microtiter plates, 20 ml of this antigen was mixed with 20 ml of serum diluted 1/100 in normal saline containing 0.78% mercapto– ethanol. Plates were kept at room temperature for 3 h. Test results were read as negative (compact blue dot) or positive (diffuse blue mat). Furthermore, all positive

2.3. Data analysis Clinical data were entered as a simple categorical data in a dbase file (dbase IV plus). FAST results were recorded as a simple qualitative categorical data. DAT results were recorded as discrete variables of simple integers as described

Table 1 Summary of clinical, serological and parasitological findings Villages

No. No. No. No.

of of of of

subjects examined sera screened subjects with hepato-splenomegaly subjects suspected as VL cases*

No. of sera tested (No. positi6e) FAST DAT No. of patients subjected to LN aspirates (No. confirmed)

Galga

Goinada

660 641 47 20

915 898 24 7

Outpatients (other villages)

All villages

50 48 45 37

1625 1587 116 64

20 (6) 12 (6)

7 (2) 8 (2)

37 (18) 38 (18)

64 (26) 58 (26)

5 (4)

2 (2)

18 (12)

25 (18)

* Defined with splenomegaly and fever of at least 2 weeks duration.

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above. Analysis was made using EPI INFO ver 5.01b (CDC/Atlanta, WHO/Geneva, 1991) and SPSS 7.5 for Windows (SPSS Inc., 1996). Statistical differences between groups of patients and study subjects were compared using uncorrected  2 which was available within the EPI Info package. Sero-prevalence rates in males and females were compared by odds ratio (OR) and the respective  2 and 95% confidence interval.

3. Results A total of 116 subjects were found to have had an enlarged spleen and/or liver. Of these, 64 were considered as VL suspects based on the presence of fever for at least 2 weeks in duration (Table 1). In the field, sera of 58 VL suspects were tested using DAT, of which 26 tested DAT positive including six cases with confirmed history of previous treatment. Aspirates of lymph nodes were made from 19 previously untreated (defined as VL cases) and three previously treated patients (defined as relapse or unresposive cases). The remaining four patients (three previously treated VL subjects and one untreated), could not be aspirated as they failed to report at the schedules and were referred to the District health centre. Three other patients were also aspirated without DAT results, as we ran out of DAT antigen after testing 58 sera. Thus, the total number of aspirated patients was 25 (22 patients with positive DAT and three patients without DAT results, Table 1). Eighteen individuals were confirmed as VL patients by demonstration of amastigotes either in smear (seven patients) or culture (two patients) or both smear and culture (nine patients). Sixteen out of 22 (72.7%) smears were positive. The figure was 78.9% (17 out of 19) when previously treated VL cases were excluded. Eleven out of 22 (50.0%) cultures grew promastigotes (52.6% when three previously treated VL cases were excluded). When smear and culture results were combined, the percent positive was 81.8% (84.2% when treated VL cases are excluded). Nine cultures were contaminated. Twelve of the 18 parasitologically confirmed VL patients belonged to the 50 subjects

who made an outpatient visit. The other six patients were detected in a total of 1575 subjects who came from Galga and Goinada. Two of the latter were previously treated VL patients. One was a child diagnosed as a VL patient 3 months perviously. It was discovered that this patient had not completed treatment. The second patient apparently was a relapse. It was reported that relapse had occurred in 7.4% of treated VL patients in this focus (Berhe et al., 1994). In previously untreated subjects, the overall sero-prevalence of anti-leishmanial antibodies detected by FAST was 4.5% (5.0% in Galga, 2.5% in Goinada and 40.0% in outpatients, Table 2). FAST was positive in 70.6% of previously treated VL patients (Table 2). Sero-prevalence rate in patients with and without symptoms of VL is depicted in Table 3. There were 8 to 19 times more asymptomatic sero-positive subjects than symptomatic sero-positive cases in Galga and Goinada; in both villages, less than one-fifth of symptomatic subjects were sero-positive (18.8 and 16.7%, respectively). In contrast, in the out-patient group, 50.0% of symptomatic patients were sero-positive. In half of asymptomatic sero-positive subjects, antibody titres were \ 6+ in DAT titre scales. In these subjects, a leishmanin skin test was positive in 25 out of the 30 tested (data not shown), indicating the development of cellular immunity during asymptomatic infection. Including previously treated VL patients, a total of 166 sera tested FAST positive. These sera were fully titrated using DAT. The distribution of titres of these sera is depicted in Fig. 1, with only one serum testing DAT negative. With the exception of one serum sample, the DAT titres of 166 FAST positive sera were equal to or above DAT titre scales of 4. Sera with large agglutination mats of FAST showed a tendency for higher titres of DAT (Fig. 1). This was also true when data was examined separately for treated VL and untreated subjects (data not shown). The distributions of DAT titres obtained in the field vis-a`-vis the different scales of FAST and parasitological findings are shown in Table 4. All 26 FAST positive sera tested DAT positive with a titre scale of at least 4. All 18 confirmed VL patients tested 3+ on FAST and 11+ on DAT.

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Table 2 Seroprevalence of anti-leishmanial antibodies based on FAST shown by village and previous history of treatment for VL Village

Past history

Number tested

Breakdown of sero-positive cases in different scales of FAST 1+

2+

3+

No. (%) sero-positive

Galga

A B Total

564 77 641

12 17 29

7 17 24

9 25 34

28 (5.00) 59 (76.6) 87 (13.6)

Goinada

A B Total

840 58 898

12 12 24

5 9 14

4 16 20

21 (2.50) 37 (63.8) 58 (6.5)

Outpatients

A B Total

40 8 48

0 0 0

2 1 3

14 4 18

16 (40.0) 5 (62.5) 21 (43.8)

All villages

A B Total

1444 143 1587

24 29 53

14 27 41

27 45 72

65 (4.50) 101 (70.6) 166 (10.5)

A, no history of VL; B, previously treated VL patients. FAST agglutinations were scored from 1+ to 3+ as follows: 3+=large agglutination mat, 2+ = small agglutination mat and 1+ =shrunken agglutination mat lacking haziness. Table 3 Sero-prevalence of anti-leishmanial antibodies in subjects with and without symptoms of VL Village

Asymptomatic group

Symptomatic group

All subjects

No. tested

No. (%) positive

No. tested

No. (%) positive

No. tested

No. (%) positive

Galga Goinada Outpatients

548 834 8

25 (4.6) 20 (2.3) 0 (0.0)

16 6 32

3 (18.8) 1 (16.7) 16 (50.0)

564 860 40

28 (5.0) 21 (2.4) 16 (40.0)

All subjects

1390

45 (3.2)

54

20 (37.0)

1444

65 (4.5)

Table excludes all individuals with past history of treatment for VL. Sero-prevalence is based on FAST antibodies.

In four out of 22 DAT positive patients, we could not show Leishmania parasites in aspirates of lymph node. The titres of these sera were 1/3200 (DAT scale of 5) in two patients and \ 1/102,400 (DAT scale of 11) in two other patients using both liquid antigen of IPB and freeze-dried DAT antigen of KIT. In Galga and Goinada, twice as many males as females were seropositive in FAST. This difference was statistically significant (OR = 2.0; Cornfield 95% confidence limits= 1.2 – 3.5; uncorrected  2 = 7.1; P B0.01). The prevalence of antibodies is clearly age dependent (Fig. 2) with an increased prevalence of FAST antibodies in

older ages. This age profile of serology probably shows the pattern of life time exposure and herd immunity rather than patterns of recent infections. The mean age of VL patients in these villages was 7.6+ (5.9) cf. the mean age of 17.0+ (5.9) in patients from the 50 self-reporting cases.

4. Discussion In this study, the prevalence of active VL was found to be 2.5/1000 (4.5/1000 in Galga, 1.1/1000 in Goinada). Based on FAST, the sero-prevalence

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Fig. 1. A chequer-board layout of DAT titres of 166 FAST positive sera shown in FAST scales (1 + , 2 +, 3 + ). DAT titres are expressed in discerete numerical scales of small integers 0 –10, for titres of 1/100 to a high of 1/102,400. Titres higher than 1/102,400 are assigned a scale of 11 and titres B1/100 are assigned a scale of −1. FAST agglutination was scored from 1 + to 3+ as follows: 3+ = large agglutination mat, 2 + = small agglutination mat, and 1 + =shrunken agglutination mat lacking haziness. , Sero-positive cases; , parasitologically confirmed sero-positive cases. Table 4 Summary of serological and parasitological findings in 64 subjects with clinical suspicion of VL No. of suspects in different scales of FAST

No. positive by DAT

Distribution by DAT titres

Parasitolgy

1

5

6

8

11

No. examined

No. positive

FAST

No. of supects

−ve 1+ 2+ 3+ NT

36 1 4 22 1

0 1 4 22 0

– 0 0 0 1

– 0 1 1 –

– 1 2 0 –

– 0 1 1 –

– 0 0 20 –

3 – 1 21 –

0 – 0 18 –

All

64

27

1

2

3

2

20

25

18

FAST, Fast Agglutination Test; DAT, Direct Agglutination Test; NT, not tested. FAST agglutination was scored from 1+ to 3+ as follows: 3+= large agglutination mat, 2+ = small agglutination mat and 1+= shrunken agglutination mat lacking haziness. DAT titres are expressed in discerete numerical scales of small integers 0–10 for titres of 1/100 to a high of 1/102,400. Titres higher than 1/102,400 are assigned a scale of 11 and titres B1/100 are assigned a scale of −1. Cut-off titre is at 1/1600 (scale of 4) as locally adopted in Ethiopia.

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Fig. 2. Seroprevalence of anti-leishmanial antibodies measured by FAST, shown by age groups. Age groups 1 – 7 are as follows: 0–9= 1; 10 – 19= 2; 20 –29 =3; 30 – 39= 4; 40 –49 =5; 50– 59 =6; 60 + =7. Bars are percent of population tested in each age group; line shows percentage sero-positive in the different age groups.

of anti-leishmanial antibodies was determined to be 35/1000 (50/1000 in Galga, 25/1000 in Goinada). Previously, using an ELISA, the seroprevalence of anti-Leishmania antibodies in this focus was determined to be 41/1000 (Hailu et al., 1996). More recent estimates using DAT are 40/ 1000 (Hailu et al., in preparation). These observations suggest that VL in this focus is a stable endemicity. This is also supported by evidence of leishmanin skin tests and incidence of the disease in the last 10 years (Hailu et al., in preparation). In a similar ecology of VL in Kenya (Baringo District), a seroprevalence of 2.7% was reported (Schaefer et al., 1995). In this survey, we have coupled both active and passive surveillance schemes and used FAST in combination with conventional DAT and parasitological methods. In 61 suspected VL patients, 36 were excluded by FAST and DAT. The availability of FAST and DAT helped to exclude 59% of subjects with big spleen and constitutional symptoms who would have been subjected to splenic or lymph node aspiration. In 22 FAST/DAT positive VL patients subjected to lymph node aspiration, 18 (81.8%) patients were confirmed by smears and

cultures (Table 4). This is higher than the rates of 50–60% previously reported in Sudanese patients (Siddig et al., 1988; Zijlstra et al., 1992), despite the fact that lymphadenopathy is a common feature of Sudanese VL patients (Siddig et al., 1990). In this endemic area, there is widespread awareness about visceral leishmaniasis. As malaria is co-endemic, hyper-reactive malarial splenomegaly (HMS) is also prevalent. As a result, patients with VL and HMS, will normally report with complaints of enlarged spleen and with or without fever. In this setting, the availability of a diagnostic tool such as DAT or FAST is crucial. In Table 3, we have shown that out of 1390 asymptomatic individuals, 45 were DAT and FAST seropositive. We do not have any evidence to suggest that these are due to false positive reactions. On the contrary, 25 out of 30 of the 45 FAST/DAT sero-positive subjects were also leishmanin skin test positive, which suggests true exposure to Leishmania infections. DAT detects antibodies in VL patients many years after treatment (Hailu, 1990; Zijlstra et al., 1991; Oskam et al., 1999). In this respect, FAST is no different as shown in our previous report (Schoone et al., 2001) as well as in this report, where

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70.6% of 143 previously treated VL patients were sero-positive. Since DAT was first developed and improved (Harith et al., 1986, 1987, 1988), it has been in use in many endemic areas. The test is used in different profiles, either to support or confirm diagnosis. The level of its use depends mainly on the magnitude of the problem and the availability of medical facilities. In areas where patient load is high and also when it is used for screening, DAT is not yet a cost-effective option at its conventional set-up. In such settings, a quick and cheaper test is needed. A major progess of the DAT has been the development of a freeze-dried antigen (Meredith et al., 1995). However, DAT still requires an incubation of 18 h. Moreover, using 5.0 ml of DAT antigen, only six to ten serum samples can be run in one plate which continues to make the test expensive. By making DAT a qualitative test, as in the case of FAST, most of these shortcomings are overcome. For instance, 5.0 ml of FAST antigen can be used to test 225 sera in five plates. In all 18 patients in whom we could show parasites in smears and/or culture, FAST mat sizes were 3+ and all were DAT positive with titre scales \ 10 (Fig. 1). If the size of agglutination mats bears any clinical and parasitological significance, as in the case of these 18 patients, FAST will make a significant contribution to the improvement of DAT in terms of alleviating the problem of reproducibility. Further field evaluations of the test is recommended.

Acknowledgements We would like to thank the Institute of Pathobiology, in particular Dr Teshome GebreMichael, for technical, administrative and ethical arrangements of the study. The technical staff of the Institute of Pathobiology (namely, Endashaw Habte, Tesfaye Getachew, Woinshet Mekonnen, Mulugeta Gichile, Nega Negussie) are thanked for bearing with the massive field and laboratory work. The medical staff of the Karat Health Centre (namely Dr Kusito Kursha, Dagnachew Asrat, Robesha Ali, Turmo Tura) are thanked for

the effective patient care rendered in the field. This research has been supported by The Netherlands Foundation for Advancement of Tropical Research (WOTRO), AIRE Developpement (France), Research and Publications Office/Addis Ababa University, Institute of Pathobiology/Addis Ababa University and the KIT Biomedical Research, The Netherlands. Dennis Guizi and Dr Ribka Amsalu of Me´ decins sans Frontie`res (Holland) are thanked for facilitating the fieldwork and for availing drugs needed to treat VL patients.

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