DIPSTICK STRIP TEST VERSUS BONE MARROW TEST FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS IN PEDIATRIC INPATIENTS MID-EUPHRATE AREA

This study was conducted in the Mid-Euphrate Region (Al-Qadisya, Najaf and Karbala provinces) during the period between first/June 2004 to first/May 2006. Two types of laboratory tests were used in this study for the diagnosis of visceral leishmaniasis in suspected patients attending pediatric hospitals in these province’s. The patients were divided into two groups; the first group included 150 children admitted to Al-Qadisya, Najaf and Karbala hospitals and the diagnosis was made on serum samples using Dipstick test, and the results indicated the positive cases by this test were 107/150 (71.4%). The second group included 27 children suspected of having visceral leishmaniasis depending on the clinical features, and microscopical smear finding by Bone marrow examination), the results indicated that the positive cases by this test were 17/27(62.9%). Fifteen out of 17 VL patients (microscopically confirmed) were found positive by dipstick test. The test was proved to be the simplest, cheapest, and rapid for the diagnosis of VL. It is a safe, highly sensitive (88.23%) and specific (60%) and can detect the disease early. It was found that all the age groups are susceptible for the disease but those who are 13-24 months old are the most susceptible. Both sexes are susceptible, but the highest incidence of infection more males (59.3%) were affected than female (40.7%). Visceral leishmaniasis was found to be more prevalent in rural than urban populations (68.3% and 31.7%) respectively. Statistical method: Chisquare test and percentage of infection were applied. P-value 0.05 INTRODUCTION he Leishmania parasite is a protozoan belonging to the order Kinetoplastida and the family of Trypanosomatidae. The genus Leishmania includes more than 20 species. The parasite exists in two morphological forms: the non flagellated amastigote (3-5 μm in diameter) living intracellular in macrophages and monocytes of the mammalian host, and the flagellated promastigotes (15-30 μm in length), in the vector and culture. Leishmaniasis is present in Iraq and was contracted by a number of the troops involved in the 2003 invasion of the country and the subsequent occupation. Confirming Visceral leishmaniasis, is a serious health hazard disease of tropical and subtropical countries, has plagued mankind since antiquity. The main problem to control visceral leishmaniasis is to establish a definite diagnosis. Till date the demonstration of the parasite in splenic/bone marrow aspirate is considered to be the gold standard. But these invasive procedures have their own drawbacks. These tests may sometime be found to be false negative, if the parasite density is low. Hence, there is a need for an alternative approach for diagnosis of visceral leishmaniasis. The clinical signs and symptoms of visceral leishmaniasis or Kala-azar are not pathognomic. Visceral leishmainasis may be confused or shared with similar diseases such as malaria, tropical splenomegaly, schistosomiasis, and others conditions. The confirmatory diagnosis of visceral diseases relies on either the microscopic demonstration of the Leishmania parasite (L.D. bodies) in the BM aspirate, or in the peripheral blood (Buffy coat). But these and other old tests became unreliable because of their risks as pain, bleeding, low sensitivity and specificity and also may be rejected by the patients and requires skill or expertise. MATERIALS AND METHODS Patients A total of 150 clinically suspected visceral leishmaniasis patients were entered to Pediatric and Maternal Hospitals of Al-Qadisya, Najaf, and Karbala provices, during the period from June 2004 to May 2006. Most the patients were admitted to the hospitals have presented with clinical manifestations such as fever, loss of the weight, anemia, hepatosplenomegaly. Methods Dipstick strip test and bone marrow were used in this study for the diagnosis of VL patients. T MJBU, VOL 27, No.2, 2009________________________________________________________________ 72 Test area not touch Sample collection Four ml of blood was collected by vein puncture into sterile test tubes and left about 2-4 hours, then serum was collected in clean test tube and store at 20 ̊C until use. BM Technique, and Geimza Stain of BM Biopsy Smear. The BM test this is the golden classical method used for detection the intracellular amastigote stage (L.D body) in BM aspirate tested microscopically, Immunochromatographic dipstick Strip Test Method (Inbios International, Seattle, USA): Test Procedure: 1. Allow the sera and chase buffer to reach room temperature prior to testing 2. Remove the Kala-azar detect strip for VL from the foil punch. 3. Add 20 μl of the serum to the test strip in the area beneath the arrow. 4. Place the test strip into well of a 96 well microtiter plates so that the end of the strip is facing downward as indicated by the arrows on the strip. 5. Add 3 drops (150 μl) of chase buffer solution provided with the kit. 6. Read the result in 10 minutes. It is significant that the background is clear before reading the test, especially when samples have low titer of antileishmanial antibody, and only a weak band appears in the test region (T). Results interpreted after 10 minutes can be misleading. Interpretation of the Results: Positive Result: The test is positive when a control line and the test line appear in the test area as shown in Figure (1), a positive result indicates that the Kala-azar dipstick detected antibody to L. donovani. Fig 1. Positive results of dipstick strip test after added serum sample (both lines were visible). Negative Result: The test is negative when only the control line appears. A negative result indicates that the dipstick did not detect antibodies of L. donovani. Figure (2). Fig 2. Negative results of dipstick strip test after added serum samples. (test line was invisible, while the control line was visible). Statistical Analysis: Chi-sequare test and percentage of infection were applied to find the significant difference between the data. P-value < 0.05 was considered significant. RESULTS A total of 150 clinically suspected visceral leishmaniasis patients were admitted to Pediatric and Maternity Hospital of Al-Qadisya, Najaf, and Karbala, during the period from June 2004 to May 2006. Most the patients were presented to hospital with anemia, weight loss, splenomegaly, hepatomegaly, and fever. The steps of diagnosis of the disease were based on the following. Clinical Diagnosis: It depends on the peculiar signs and symptoms related to this disease such as anemia, weight loss, enlarged spleen, enlarged liver, and duration of fever. This study was showed that of the patients were suffered from anemia and enlarged spleen (Figure-3). Test Line Invisible Control line (C) Test Line (T) Control Line (T) Test area-do not touch ________________________________________________________________________________________________MJBU, VOL 27, No.2, 2009 73 Fig 3. Child of 60 months old with enlarged spleen due to VL (photo was done after the agreement of his