Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

ABSTRACT.

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

INTRODUCTION

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has a 100% mortality rate unless treated.¹ It is mainly caused by Leishmania donovani in India and Africa, Leishmania infantum in the Mediterranean area. and L. infantum syn Leishmania chagasi in Latin America.² VL is endemic in southern Saudi Arabia, as determined by prospective observations performed by Ibrahim and others.³ The latest epidemiological examination confirmed that the total number of VL cases during the period from 2007 to 2015 was 120 in the Jazan region.⁴ In 1992, VL was described in Albaha region of the southwest Saudi Arabia by Al-Jurayyan and others.⁵ Additionally, VL has been diagnosed in an area previously not considered to be affected by endemic L. donovani in Tabouk in northern Saudi Arabia.⁶ This may indicate either the formerly unrecognized presence of L. donovani or provide evidence of the changing pathogenic role of Leishmania tropica, which is the most common parasite in northern Saudi Arabia.

Clinical diagnosis of leishmaniasis is generally suggestive, and it is not usually subject to standardization. A definitive diagnosis is based on the clinical manifestations of the patient together with the detection of the intracellular stage of the parasite by examination of smears or biopsies of infected tissues and the culturing of specimens.⁷

Parasitological confirmation of VL infection depends mainly on spleen aspiration; liver, bone marrow (BM), or lymph node tissue staining with Giemsa may be accomplished, but this method varies in sensitivity (58.3%, 78%, and 81–100% for lymph node aspirates, 70.2% for BM aspirates and 96.4% for spleen aspirates).⁸ Culture of materials from the BM, lymph nodes, and spleen is useful for parasite isolation and identification, which could be neglected by direct examination of smears; however, the smear result might also be obtained weeks earlier than the parasite growth result.²

Polymerase chain reaction (PCR) technique is definitive, as it detects parasite DNA.^9,10 PCR of DNA from blood samples from VL patients detects disease with a sensitivity that is as high as 100%.¹¹ More recently, quantitative real-time PCR has emerged as the desired method.¹² Several real-time PCR techniques have been suggested with high sensitivity and specificity.^13–17

Serological tests are most commonly used, and the direct agglutination test (DAT) and rK39 test have been widely evaluated in VL endemic areas and have produced variations in specificity and sensitivity.^18–20 Although there are a number of problems associated with serological tests, most of these tests do not differentiate between current and past infections. A metanalysis assessing 30 studies employing DAT showed its high sensitivity and specificity.²¹

The diagnosis of VL in the Jazan region relies upon detecting the parasite in a BM aspirate, which is invasive, painful, and requires hospital admission and skilled clinical personnel. There is a pressing need to implement of much less invasive and more sensitive and simple techniques for the routine analysis of VL. This is the first study on diagnosis of VL evaluating the DAT and PCR in the Jazan region of southwest Saudi Arabia. The desired outcome of this work is to implement a diagnostic approach for VL that can be used in the Jazan region of southwest of Saudi Arabia that is less invasive and more accurate and can be used in the vicinity of the endemic area, which is similar to other areas in the tropics.

MATERIALS AND METHODS

Ethical approval.

The study was approved by the Jazan Directorate of Health Affairs Research Ethics Committee, King Fahad Central Hospital, registry number 042/October 2017, and the Scientific Research Ethics Committee of Jazan University, reference number 39/4‐274 December 2017. The study was performed in accordance with the ethical standards stated in the 1964 Declaration of Helsinki.

Informed consent.

Inform consent was obtained from adult participants, parent, and/or legal guardian for participants under the age of 18 years before enrollment in the study.

Study participants.

Eighty participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria group, were enrolled in this work from the Jazan area of southwest Saudi Arabia, which is a region of endemic VL, from December 2017 to October 2019. All VL-suspected patients presenting with symptoms consisting of fever, splenomegaly and/or hepatomegaly, pancytopenia, and weight loss, which are based on the WHO clinical VL case definition,²² with no records of past VL who were negative for HIV were selected as suspected VL cases. Thirty clinically healthy individuals without a previous history of VL were identified as endemic controls. Twenty patients with malaria confirmed by PCR were selected as malaria group disease controls based on clinical symptoms similarity with VL consisting of fever, low hemoglobin, and loss of weight. The participant data were collected by questionnaire. Treatment of patients with confirmed VL was performed based on national guidelines, all confirmed VL patients are shown a 100% response to treatment.

BM sample.

Collection, detection, and confirmation of outcomes were performed at different hospitals in the Jazan area, only VL-suspected patients underwent BM biopsy, whereas the DAT, and PCR were performed among the all study participants. The BM smear was used as reference method to calculate the sensitivity of the real-time PCR and the DAT.

Preparation of samples and DNA.

Five milliliters of venous blood were obtained from every suspected VL patient and control by venipuncture and collected in ethylenediaminetetraacetic acid (EDTA)-containing and plain tubes. Blood was centrifuged at 4,000/rpm for 15 minutes at 4°C for separation of the buffy coat, which was used for DNA isolation, and serum, which was used for antibody detection. The buffy coat, whole blood, and serum were transported to the molecular biology and immunology laboratories at the medical research center of Jazan University. Genomic DNA was isolated from whole blood of malaria group and buffy coat of suspected VL, and endemic control by using a QIAamp DNA Tissue and Blood Mini Kit (Qiagen, Germantown, MD) by following the manufacturer’s instructions. Inform consent was obtained from adult participants, parent, and/or legal guardian for participants under the age of 18 years before sample collection.

Real-time PCR protocol.

Real-time PCR was carried out using the ribosomal inner transcribed spacer 1 (ITS1) primers LITSR: F 5'-CTGGATCATTTTCCGATG and L. 58S: R 5'-TGATACCACTTATCGCACTT to detect the DNA of Leishmania species in blood samples as described by ElTai and others.²³ 2x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was used for real-time PCR according to the manufacturer’s instructions. In brief, 25 μL of reaction mix was prepared that contained 10 μL DNA template, 10 μL SYBR Green, 1 μmol of each primer, and 3 μL sterile water. Amplification was achieved on a 7,300 real-time PCR instrument (Applied Biosystems) using fluorescein amidites as the detector with the following reaction conditions: 94°C for 5 minutes, followed by 40 cycles of 94°C for 30 seconds, 55°C for 60 seconds, and 72°C for 60 seconds. Each run also included positive control DNA from the L. donovani complex that was characterized by an iso-enzyme as MON 82, which was generously provided by Professor Maowia at the Mukhtar Laboratory, Institute of Endemic Diseases, University of Khartoum, Sudan, and a nontemplate control (NTC). Samples were run in triplicate, and a mean cycle threshold (Ct) less than 32 represented the positive control upon comparison of the positive and negative controls (Figure 1). The primers amplified 350 bp fragments that were visualized by 1.5% agarose gel electrophoresis using the SYBR safe gel staining dye (Invitrogen, Seol, South Korea).

Figure 1.

Amplification curve of real-time polymerase chain reaction (PCR) of blood samples positive for Leishmania DNA from visceral leishmaniasis (VL) patients, with cycle threshold (Ct) values ranging from 14 to 30; no Ct value was produced by the nontemplate control (NTC). The positive Leishmania donovani control had a Ct value of 20. This figure appears in color at www.ajtmh.org.

Sequencing.

Sequencing was performed for four samples with a high concentration of DNA, which was amplified by the kDNA primer as described by Noyes and others^24,31 to produce a 720 bp fragment and by the ITS1 primer to produce a 350 bp fragment. The amplification reactions consisted of a 25 μL volume containing 10 μL exTEN 2X Ready To Use Master Mix (1st Base, Singapore), 1 μmol of each primer, 4 μL of sterile nuclease-free water, and 10 μL of DNA. DNA was amplified through PCR using the same cycling conditions mentioned earlier, and the products were sequenced commercially (Macrogen, Seoul, South Korea). The obtained sequencing result was checked by a search of BLASTn in GenBank to determine homology, to confirm the results of the PCR diagnostic method and to identify the possible Leishmania species causing VL in the study area.

Direct agglutination test.

To detect VL antibodies in sera from all study groups, we used a liquid DAT antigen (containing stained and killed L. donovani promastigotes), which was generously provided by Dr. Elharith at the Biomedical Research Laboratory, Ahfad University for Women, Sudan. The test was performed according to an advanced protocol defined previously.^25,26 Briefly, the semiquantitative test was performed in a V-shaped microtiter well plate, in which a serial dilution of the serum was mixed with the antigen, and positive sera from VL patients from Sudan and negative sera were included in each test. The sera were tested by using 2-fold serial dilutions (from 1:200 to 1:200,800). Serum samples showing titers of 1:3,200 or higher were considered VL-positive.^27,28 The test results were read after overnight incubation at 25°C, and two trained technicians read and reported the test results blindly to avoid bias due to subjectivity during the titer reading. For a positive test result, the well appears cloudy, and for a negative test result, the reagent is deposited at the lowest point of the well (Figure 2).²⁹

Figure 2.

Direct agglutination test (DAT) result: A1-12: negative control samples, B1-12: positive control sera from VL patients with titers of 1:204,800, as indicated by the red line, C1-12 and D1-12: positive VL samples, E1-12: negative samples with a titer of 1:200, F1-F12: endemic control samples with a titer of 1:800, G1-12: disease control samples, and H1-12: positive VL samples. This figure appears in color at www.ajtmh.org.

Statistical analysis.

Data analysis involved descriptive variables in addition to inferential data. Simple tabulation and determination of frequencies were applied to afford a general assessment of the statistics. Numerical statistics are presented as the mean and SD. Student’s t test was used to compare numerical data, whereas Fisher’s exact test was used to evaluate differences in proportions. The sensitivity of real-time PCR and the DAT were calculated among suspected VL patient according to the gold standard (BM smear) and supported by means of 95% CIs. The proportions of the positive and negative results of real-time PCR and DAT performed for the analysis of Leishmania infection were compared using the 2-tailed McNemar’s test for matched pairs.³⁰ A P value < 0.05 was considered significant. The analysis was performed in compliance with the Standards for Reporting Diagnostic Accuracy (STARD) statement.³¹

RESULTS

The demographic and clinical data from the study participants and the frequency of VL infection among suspected VL patients shown in Tables 1 and 2, respectively.

Table 1.

Demographic and clinical characteristics of all study participants

Vaiable	N			%
Gender
Male	62			77.5
Female	18			22.5
Age groups
< 15 years	23			28.8
15–44 years	46			57.5
> 45 years	11			13.8
Nationality
Non-Saudi	20			25.0
Saudi	60			75.0
Symptoms	Suspected VL group	Malaria group	Control group
Splenomegaly Absent	07	20	30	71.25
Present	23	00	00	28.75
L. Node Enlargement Absent	27	00	00	96.25
Present	03	00	00	03.75
Fever absent	00	00	30	37.5
Present	30	20	00	62.5
Hemoglobin low	28	16	00	55.0
Normal	02	04	30	45.0
Total	80			100

Table 2.

Prevalence of Leishmania infection among suspected VL patients

	Suspected VL patients
Variable	N-positive/ N-tested	% (+ve)	P Value Fisher exact test
Gender			0.525
Male	(7/23)	30.0
Female	(3/7)	42.9
Age groups			0.188
< 15 years	(7/11)	63.6
15–44 years	(1/12)	8.3
> 45 years	(2/7)	28.1
Nationality			0.102
Saudi	(5/22)	22.7
Non-Saudi	(5/8)	62.5
Splenomegaly			0.077
Absent	(0/7)	0
Present	(10/23)	45
L. Node Enlargement			0.142
Absent	(0/20)	0
Present	(3/10)	33.3
Overall	(10/30)	33.3

VL = visceral leishmaniasis.

Results of BM smear.

BM smear was confirmed positive in 10 suspected VL patients out of 27 tested with 37% frequency. Three samples were missed confirmation of BM smear result for this reason the total of 27 out of 30 individuals result were included and analyzed.

Results of real-time PCR.

Leishmania parasite DNA was amplified by real-time PCR from the blood of 10 suspected VL patients out of 27 tested with 37% frequency, 100% sensitivity, and a Cohen’s kappa value of 1 (Table 3). Parasite DNA was not detected in the endemic and malaria group and control samples. The real-time PCR results were supported and confirmed by DNA sequencing.

Table 3.

Comparison of the sensitivity of real-time PCR and DAT, with BM smear as reference method for the diagnosis of VL infection expressed as percentages with the 95% CI

Measure	Real-Time PCR (n = 27)				DAT (N = 27)
	%	N-positive/N-tested		(95% CI)	%	N-positive/N-tested		(95% CI)
Sensitivity	100	(10/27)		(72.25, 100)	100	(10/27)		(72.25, 100)
Cohen’s kappa	1			–	0.922			–

CI = confidence interval; DAT = direct agglutination test; PCR = polymerase chain reaction.

Results of the DAT.

The DAT-detected antibodies against Leishmania in 10 suspected VL serum samples out of 27 tested with 37% frequency, 100% sensitivity, and a Cohen’s kappa value of 0.922 (Table 3). One false positive sample with high antibody titer was detected by DAT, which was negative according to both the BM smear and real-time PCR. No significant antibody titer for VL was detected in any of the endemic and disease control samples.

Results of sequencing.

The sequencing results of four of the PCR-amplified products produced by the ITS1 and kDNA primers, identified three L. donovani and one L. infantum species followed submission of the sequences to GenBank as Leishmania species causing VL in the study area are shown (Table 4). All the four patients were male, adult children, Saudi nationality, and borne and lived in Jazan area.

Table 4.

Identified Leishmania species followed submission of the sequences to GenBank

Samples	ITS1 primer		kDNA primer		Identified species
	Identity% with similar species on GenBank	Accession number	Identity% with similar species on GenBank	Accession number
VL9	99.64	MN244151.1	99.65	CP029526.1	Leishmania donovani
VL11	100	KM677134.1	99.65	XR_001203206.1	Leishmania infantum
VL13	99.64	MN244151.1	99.65	CP029526.1	L. donovani
VL14	99.64	MN244151.1	99.62	CP029506.1	L. donovani

ITS1 = inner transcribed spacer 1.

DISCUSSION

Visceral leishmaniasis is endemic to the Jazan region of southwest Saudi Arabia and is a fatal disease, an early and accurate analysis is crucial. This is the primary study performed within the region that has focused on VL diagnosis.

In this study, we assessed the sensitivity of real-time PCR and the DAT with blood samples gathered from the Jazan area of southwest Saudi Arabia from 2017 to 2019. The evaluation of the diagnostic tools used for VL inside the study area is critical because the burden of VL remains a major issue for the general public health, as it affects mainly infants and young children.

In this study, the high sensitivity of real-time PCR of Leishmania parasite DNA for all clinically suspected VL samples ensured that the diagnosis of VL based on blood samples was achieved by real-time PCR, and similar results were reported previously.^11,32,33 The real-time PCR-positive sample results were supported and confirmed by sequencing. Parasite DNA was not detected in any control samples, indicating the high specificity of the molecular techniques, which was similar to the findings described by Hossain and others.³³

To focus on Leishmania species causing VL in the study area, we sequenced four of the DNA products amplified by the kDNA and ITS1 primers. The PCR products for both target genes identified L. donovani and L. infantum following submission of the sequences to GenBank. Both species were isolated and identified by iso-enzyme method before from human and wild animals in the study area.^3,34 Similar findings of isolated and identified L. donovani and L. infantum species were reported from Yemen³⁵ a shared border country to the study area.

The DAT-detected antibody against Leishmania in all clinically suspected VL samples with 100% sensitivity. Our findings were in agreement with those of 30 studies performed before that confirmed the high sensitivity and specificity of the DAT.²¹ No significant antibody titer against VL was detected in any of the endemic and disease control samples, although one false positive sample was detected by the DAT among suspected VL patients, which was negative according to both the BM smear and real-time PCR. The final diagnosis of this patient indicated an autoimmune hepatitis disease, so the case definition of the patient’s immune status had to be clarified before applying the test, although some recent studies have highlighted that the DAT shows ideal sensitivity for the diagnosis of VL-/HIV-coinfected patients and is superior to various serological assessments.^36,37

The DAT is a simple, rapid method that makes use of a less invasive method and has been verified by field studies. The reported sensitivities in this study of the DAT justify the set of guidelines proposed for VL diagnosis in the Jazan region, where the DAT is first used for the diagnosis of suspected VL cases and can be complemented with parasitological tests to ensure an accurate analysis.³⁸

To our understanding, this is the first evaluation of real-time PCR and the DAT for VL diagnosis in the Jazan area. The outcomes of this study can inform the directorate of health affairs and the health practitioners within the study area regarding the strengths and challenges of serological diagnosis; however, PCR diagnosis should always be taken into consideration as a confirmatory test for infection and as a suitable diagnostic solution for VL.

CONCLUSION

The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

Note: Supplemental material appears at www.ajtmh.org.