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Comparative study of hospitalized patients of suspected visceral leishmaniasis in Basrah, Maysan and Thi-Qar governorates by used dipstic rk39 test


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Comparative study of hospitalized patients of suspected visceral leishmaniasis in Basrah, Maysan and Thi-Qar governorates by used dipstic rk39 test



Hind Mahdi Jarallah Abdul-Hussein H. Awad

Marine science center, Univ. Basrah Coll. Educ. Univ. Basrah

Summary

The present study includes a comparative study among 3 governorates concerning the dipstick rk39 test. Ninety suspected cases of VL were collected from admitted patients to Basrah, Maysan and Thi-Qar hospitals based on the clinical features (sings and symptoms). These 90 cases were: (30 suspected cases collected from Basrah General Hospital and Basrah Maternity and children Hospital in Basrah governorate, 30 suspected cases collected from Al-Sadr General Hospital in Maysan governorate and 30 suspected cases collected from Bint-Al-Huda Hospital in Thi-Qar governorate). The positive cases with dipstick rk39 test out of 90 suspected cases of VL were 76(84.44%) as follows: (24(80%) in Basrah hospitals, 27(90%) in Maysan hospital and 25(83.33%) in Thi-Qar hospital), 52(68.42%) male and 24(31.57%) female. Statistically, there are no significant differences p>0.05 among 3 governorates by utilized dipstick rk39 test.



Key words: Visceral leishmaniasis, dipstic rk39 test.
Introduction

Visceral leishmaniasis caused by geographic variants of the Leishmania donovani complex (L. donovani, L. infantum, L. chagasi), is a progressive wasting disease of dog and humans that is often fatal if untreated (Baneth, 2006). Visceral leishmaniasis is, also known in Asia as black fever or kala-azar, and black disease, dum-dum (WHO, 1998). The incubation period from the time of the exposure to symptoms is 10 days to more than 2 years but is usually 3-8 months (Davidson, 1999). Kala-azar is a Hindu name given to this disease which was characterized by a blackening of the skin on the forehead and hands. L. donovani complex organisms parasitize reticuloendothelial cell (RE), however, they are not confined to histocytes in the mucous membrane or subcutaneous tissues but parasitize RE cells throughout the body (Garcia and Bruckner, 1993). Visceral leishmaniasis (VL) is one of the most important endemic diseases in Iraq and is known to occur since (1916) when first infection with Leishmania was recorded (Kadhim et al., 1978 ; Sukkar, 1978). In Iraq, the disease was found to be more prevalent in the central region and mostly in the rural village, area and semi-desert region (Kadhim et al., 1978 ; Sukkar, 1978). In South of Iraq the main focus governorates of VL in few last year is (Maysan, Thi-qar, Basrah and Al- Muthana) (Sukkar, 1996). Iraqi southern marshes which form a triangle region bound by three major southern cities, Thi-Qar to the west, Maysan to the northeast and Basrah to the south. The three major marshes: Al-Hammar, The Central marshes and Al-Huwaiza marshes form the core of the marshlands of southern Iraq (Bedair et al., 2005). The people in the villages of marshlands is famous with fishing and hunting birds, the shrubs covered most the region of this area and there are many cattle, sheeps, buffaloes, birds, dogs and some wild animals like jackals that live in the border of the villages. The wide range of vegetation, the humidity, shrubs and old mud houses is regarded as a suitable environment for huge number of sand flies . the aim of this study Is to Comparing evaluate the sensitivity of dipstick rk39 test for diagnosis visceral leishmaniasis patients in Basrah, Maysan and Thi-Qar hospitals.


Materials and methods

Sample collection

Sera were collected from suspected VL patients admitted to Basrah governorate hospitals (Maternity and children Hospital and Basrah General Hospital, 30 cases), from Maysan governorate Hospital (As-Sadr General Hospital, 30 cases) and from Thi-Qar governorate Hospital (Bent- Al-Huda Hospital, 30 cases). These sera were used for comparative study among three governorates by using serological dipstick rk39 test. The clinical features were collected from each hospitalized VL patient, include fever, anaemia, cough, vomiting, loss weight, paleness, jaundice, diarrhea, pallor, abdominal distention, loss of appetite and hepatosplenomegaly . The suspected patients were classified according to some information collected from their parents.



Immuno-chromtography Dipstick strip Assay

1- Kit contents :

Kala-azar dipstick test strips are a membrane, pre-coated with a recombinant VL antigen on the test line region and chicken anti-protein A on the control line region. The kit contains twenty – five (25) individually pouched test strips and one vial of chase Buffer solution. The sealed pouch containing the test strip along with buffer vial is designed to be stored at room temperature (20 -28°C) for the duration of its shelf life. Exposure to temperatures over 30°C can impact the performance of the test and should be minimized . The strips should not be frozen . The test should be used within one hour after removal from the foil pouch to prevent exposure to humidity.



2- Test procedure

  1. The sera and chase buffer were allowed to reach room temperature prior to testing.

  2. The kala-azar dipstick strip removed from the foil pouch.

  3. 20µl of serum added to the test strip in the area beneath the arrow.

  4. The test strip placed into a test tube so that the end of the strip is facing downward as indicated by the arrows on the strip.

  5. 2-3 drops (150µl) of the buffer solution provided with this test kit were added.

  6. The results read in 10 minutes. It is significant that the background is clear before reading the test, especially when samples have low titer of anti-leishmanial antibody.

Note: The validity of the dipstick rk39 test strips was checked on 10 confirmed visceral leishmaniasis cases.

3- Read result:

A- Positive result: The test is positive when a control line and test line appear in the test area (red line in both regions). A positive result indicates that the kala-azar dipstick detects antibodies to members of L. donovani complex .

B- Negative result : The test is a negative result when only the control line appears (red line in control region only). A negative result indicates that the kala-azar dipstick detected did not detect antibodies to members of L. donovani complex.

C- An-Invalid result : If no lines appear at either the control or test line areas the test is invalid. The test is also invalid if no control line appears, even though a test line is seen. It is recommended result using a new or (fresh) dipstick test and fresh serum.

Results

This study includes (90) patients children (30 admitted to Maternity and children Hospital and General Hospital in Basrah, 30 patients admitted to As-Sadr General Hospital in Maysan and 30 cases of children patients from Bint-AL-Huda Hospital in Thi-Qar. It was found that 76(84.4%) was positive cases with rk39 dipistick test Table(1). There are no significant differences in this test among 3 governorates. All these patients have the main clinical features of VL such as (fever, anemia, pale and splenohepatomegaly)



Table (1): Comparison of sero-positive cases of VL patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar

Governorate

No. of cases

No. of +ve cases

%

Basrah

30

24

80

Maysan

30

27

90

Thi – Qar

30

25

83.33

Total

90

76

84.44

χ2= 0.625 df= 2 p>0.05

1- Age and sex distribution

Table(2, A, B, C) and Figure(1) show the positive cases of each governorate distributed according to the age and sex. Maysan shows the highest percent with dipstick test 27(90%) when compared with Basrah and Thi-Qar, 24 (80%) and 25(83.33%) respectively.



The positive cases with dipstick in males were (87.5, 70.37, 48%) in Basrah, Maysan and Thi-Qar governorates while in females (12.5, 29.62, 52%) respectively. Statistically, There are significant differences (p<0.05) in all ages groups and between males and females among 3 governorates. The positive cases of male and females in all 3 governorates were (68.42, 31.57%) respectively with significant differences( p<0.05) Table (3).

Table (2) Age and sex distribution of sero-positive cases of VL patients diagnosed with dipstick test in Basrah (A), Maysan (B) and Thi-Qar (C) governorate

A

Age

years

Basrah

Total

Sex

No.

%

Male

Female

No.

%

No.

%

<1

7

77.77

2

22.22

9

37.5

1

5

83.33

1

16.66

6

25

2

5

100

0

0

5

20.83

3

2

100

0

0

2

8.33

4

1

100

0

0

1

4.16

5

1

100

0

0

1

4.16

Total

21

87.5

3

12.5

24

100

χ2= 57.76 df= 1 p<0.05



B

Age

years

Maysan

Total

Sex

No.

%

Male

Female

No.

%

No.

%

<1

8

66.66

4

33.33

12

44.44

1

5

62.5

3

37.5

8

29.62

2

3

75

1

25

4

14.81

3

2

100

0

0

2

7.40

4

1

100

0

0

1

3.70

5

0

0

0

0

0

0

Total

19

70.37

8

29.62

27

100

χ2= 16.6 df= 1 p<0.05



C


Age

years

Thi – Qar

Total

Sex

No.

%

Male

Female

No.

%

No.

%

<1

6

60

4

40

10

40

1

2

25

6

75

8

32

2

1

50

1

50

2

8

3

2

66.66

1

33.33

3

12

4

1

50

1

50

2

8

5

0

0

0

0

0

0

Total

12

48

13

52

25

100

χ2= 0.16 df= 1 p>0.0



Table (3) Age and sex distribution of sero-positive cases of VL patients diagnosed with dipstick test in all 3 governorates

Age

years

Sex

Total

Male

Female

No.

%

No.

%

No.

%

<1

21

67.74

10

32.25

31

40.78

1

12

54.54

10

45.45

22

28.94

2

9

81.81

2

18.18

11

14.47

3

6

85.71

1

14.28

7

9.21

4

3

75

1

25

4

5.26

5

1

100

0

0

1

1.31

Total

52

68.42

24

31.57

76

100




χ2= 26.22 df= 4 p<0.05

Figure (1): Age distribution of positive cases of visceral leishmaniasis patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar governorates

2- Clinical features

Fever, pale, anemia, weight loss, vomitin, cough splenomegaly and hepatomegaly were the most commonly reported features. Statistically there are significant differences (p<0.05) in clinical features Table (4).



Table (4) Clinical features of sero-positive cases of VL patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar

Clinical Features

Basrah

Maysan

Thi – Qar

Total

No.

%

No.

%

No.

%

No.

%

Fever

24

100

27

100

25

100

76

100

Anaemia

24

100

27

100

25

100

76

100

Splenomegaly

15

62.5

23

85.18

20

80

58

76.31

Hepatomegaly

7

29.16

15

55.55

9

36

31

40.78

Pale

9

37.5

16

59.25

10

40

35

46.05

Cough

12

50

12

44.44

13

52

37

48.68

Loss of appetite

1

4.16

3

11.11

1

4

5

6.57

Loss of weight

5

20.83

7

25.92

8

32

20

26.31

Diarrhea

3

12.5

1

3.70

1

4

5

6.57

Vomiting

0

0

2

7.40

1

4

3

3.94

Jaundice

2

8.33

4

14.81

4

16

10

13.15

Abd. Distention

7

29.16

9

33.33

5

20

21

27.63

Dyspenea

0

0

1

3.70

0

0

1

1.31

χ2= 86.10 df= 12 p<0.05

3- Environmental variables

Table (5) demonstrated the numbers and the percentages and the presence or the absence of these variables Sand flies were reported in the environmental of all cases of each governorate (100%). The percentage distribution of dog reaches to (100%) in Maysan and Thi-Qar, while in Basrah it reaches to (79.16%). There are significant differences (p<0.05) among environmental variables in all 3 governorates.



Table (5) Distribution of environment variable of sero-positive cases of VL patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar

Variables

Basrah

Maysan

Thi – Qar

Total

No.

%

No.

%

No.

%

No.

%

Sand fly

24

100

27

100

25

100

76

100

Stray dogs

19

79.16

27

100

25

100

71

93.42

Domestic animals

13

54.16

24

88.88

18

72

55

72.36

Rodents

16

66.66

27

100

23

92

66

86.84

Wild canine

2

8.33

8

29.62

5

20

15

19.73

χ2= 56.20 df= 4 p<0.05

4- Type of building materials

This study includes the distribution of 90 cases of VL from 3 governorates according to the type of building materials Table (6).

There are significant differences (p<0.05) among type of building materials for 3 governorates.

Table (6) Distribution of building type materials of sero-positive cases of VL patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar


Type of building material

Basrah

Maysan

Thi-Qar

Total

No.

%

No.

%

No.

%

No.

%

Reed

0

0

2

7.40

3

12

5

6.57

Mud

1

4.16

6

22.22

3

12

10

13.15

Others

23

95.83

19

70.37

19

76

61

80.26

Total

24

100

27

100

25

100

76

100

χ2= 36.6 df= 2 p<0.05

5- Parents education level

In this study the level of the parents education for each patient was recorded in Basrah, Maysan and Thi-Qar governorates Table (7A,B).



Statistically, there are significant differences (p<0.05) among the level of education for the father and the mother in all 3 governorates.

Table (7, A) Paternal (father) education level of sero-positive cases of VL patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar governorates

Level education

Basrah

Maysan

Thi – Qar

Total

No.

%

No.

%

No.

%

No.

%

Illiterate

3

12.5

15

55.55

8

32

26

34.21

Primary & intermediate

15

62.5

10

37.03

12

48

37

48.68

Secondary& High Education

6

25

2

7.40

5

20

13

17.10

Total

24

100

27

100

25

100

76

100

χ2= 17.28 df= 2 p<0.05

Table (7, B) Maternal (mother) education level of sero-positive cases of VL patients diagnosed with dipstick test in Basrah, Maysan and Thi-Qar governorates

Level education

Basrah

Maysan

Thi – Qar

Total

No.

%

No.

%

No.

%

No.

%

Illiterate

10

41.66

20

74.07

13

52

43

56.57

Primary& intermediate

12

50

7

25.92

10

40

29

38.15

Secondary& High Education

2

8.33

0

0

2

8

4

5.26

Total

24

100

27

100

25

100

76

100

χ2= 20.87 df= 2 p<0.05

Discussion

In this study the dipstick is used as a tools which is reliable, easy, fast and simple for the diagnosis of VL. The strip testing proved simple to perform and yielded results within five minutes. The diagnosis of VL in Basrah hospitals by bone marrow examination, but that test is difficult and needs to specialized doctors. The parents of the children do not favor and refuse this method. It was demonstrated that bone marrow examination was not a practical method and gave higher negative results (Zijlster et al., 1998). On the other hand, stained direct smear examination is also time consuming and requires experts to detect amastigote stage. This study includes 90 cases of suspected VL patients collected from hospitals of Basrah, Maysan, and Thi-Qar governorates (30 patients from each governorate) were selected to a comparison study using dipstick rk39 test as a serological test. This comparison study is regarded as a first attempt by utilized serological diagnostic method tests among suspected VL patients who belong to hospitals of Basrah, Maysan and Thi-Qar governorates.

In Basrah, Maysan and Thi-Qar patients group, out of 90 suspected VL cases the sero-positive cases with dipstick were 76(84.4%) the highest rate was found in Maysan 27(90%),then Thi-Qar group, 25(83.3%) and the last was in Basrah 24(80%). Gani (2006) reported that dipstick test in Basrah was positive only in 3(2.0%) out of 146 cases of VL. Sundar et al. (2000) reported a 100% sensitivity for the nitrocellulose paper impregnated the rk39 antigen test using peripheral blood samples from Indian patients. Bern et al. (2000) concluded that the rk39 test is comparable to Parasitological test in terms of their sensitivity and can replace it as the basis for a decision to treat VL at peripheral health centers in endemic areas. The present study demonstrated that there are significant differences between males and females of positive cases of VL patients diagnosed with dipstick test in all 3 governorates.

Seventy six out of 90 cases of suspected of kala-azar found in this study fever, anemia, and splenomegaly were the predominant clinical features, these signs and symptoms are the same as those found in other clinical studies in Iran (Mohebali et al., 2006). In all 76 positive cases of VL with dipstick test have sand flies 100% in their houses with high density of stray dogs are present around the houses of the patients in all 3 governorates. El-Edan (2001) showed that the presence of domestic animals in the houses of the patients play an important role in providing blood meals for sand fly vector. There were significant differences between sero-positive and the type of the building material among 3 governorates. The human component such as behavioral activities or houses design have been identified as factors for leishmaniasis (Davies et al., 2000).

The present study shows a significant differences between father and mothers level of education in 3 governorates of the present study; therefore, the parents education is plays an important role to improve the socioeconomic status and the standard of living, and this may confirm the importance of the health education in the control program of VL. It was concluded from the present study that developing a more specific Leishmania dipstick test should be pursued, there are many potential advantages of such a tool with respect to other diagnostic methods, a large number of samples can be processed quickly and with minimum effort. This study showed that the dipstick rk39 test, is simple, faster, sufficiently sensitive in the diagnosis of active VL in human.

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