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Am. J. Trop. Med. Hyg., 79(6), 2008, pp. 839-842
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Detection of Leishmania infantum Kinetoplast DNA in the Whole Blood of Asymptomatic Individuals by PCR-ELISA and Comparison with Other Infection Markers in Endemic Areas, Southern Iran

Abdolvahab Alborzi*, Bahman Pourabbas, Frahad Shahian, Jalal Mardaneh, Gholam R. Pouladfar, AND Mazyar Ziyaeyan
Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Visceral leishmaniasis (VL), caused by Leishmania infantum, is endemic in southern Iran. To detect asymptomatic individuals, we used kinetoplast DNA (kDNA) polymerase chain reaction (PCR)-ELISA methods on 388 blood samples of healthy persons in two endemic loci and compared the results with the leishmanin skin test (LST) and the immunofluorescent antibody test (IFAT). kDNA PCR, LST, and IFAT were positive in 95 (24.5%), 132 (34%), and 212 (54.6%) cases, respectively. Fifty-five (21.4%) individuals that were LST negative were PCR positive. All PCR-positive individuals had a titer of ≥ 1:20, whereas 45% of those that were IFAT positive were PCR positive. For a reliable index of prevalence rate of infection, LST alone is not sufficient and needs to be accompanied by PCR-ELISA. The high rate of kDNA-positive results may indicate the possibility of humans being a reservoir and source of transmission. In endemic areas, kDNA PCR-ELISA is not a reliable test for the diagnosis of active VL.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Visceral leishmaniasis (VL; kala-azar), a vector-borne zoonotic disease transmitted by sand fly bites, is endemic in rural and tribal areas of southern and northwestern Iran.1 Leishmania infantum is known to be the dominant Leishmania strain in Iran, which mainly affects children.1,2 Kala-azar is characterized by fever, hepatosplenomegaly, neutropenia, hyperglobulinemia, and anemia with or without lymphadenopathy. Those infected with the viscerotropic Leishmania species may, however, remain asymptomatic, which is the most common outcome of infection in endemic areas.3

During the last few years, epidemiologic reports in Brazil, Spain, and France have shown that a considerable number of parasite DNA were found in the blood of asymptomatic individuals.3–6 The mini-circle kinetoplast DNA (kDNA) has been used as a sensitive and specific target for selective amplification of parasite DNA in various studies.7–10 The poly-merase chain reaction (PCR)-ELISA technique for the detection of L. infantum mini-circle kDNA has been shown to be highly sensitive and specific, even when peripheral blood samples are used.4,11

The aim of this study was to investigate the persistence of L. infantum kDNA in healthy individuals living in endemic areas and compare the results with the leishmanin skin test (LST) and the immunofluorescent antibody test (IFAT).


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Population studied. A cross-sectional study was performed on 388 blood samples from randomly selected healthy persons living in the two areas where the disease is endemic (Ghir Karzin and Sar Mashhad) from August to September 2005. All participants were screened by a trained physician. Cases who had fever, hepatosplenomegaly, or history of cutaneous or visceral leishmaniasis were excluded from the study. The participants were divided into three groups in terms of their ages: Group A (N = 106), 1–5 years; Group B (N = 122), 6–15 years; Group C (N = 160), 16–35 years. This study was conducted with the approval of the Ethics Committee of Shiraz University of Medical Sciences, Iran, and informed consents were obtained from all individuals or their guardians.

Leishmanin skin test. All participants underwent an LST by injecting 0.1 mL of the leishmanin antigen (Leishmanin; Pasteur Institute of Iran) intradermally on the volar aspect of the left forearm. Control consisted of 0.1 mL of leishmanin diluent injected into the other forearm. LST was considered positive if, 48 hours after injection, a 5-mm induration or more was found at the test site without induration at the control site.

Sample collection. Two milliliters of peripheral blood collected in EDTA-coated tubes was aliquoted and frozen at –70°C for PCR study. At the time of sampling, the serum for IFAT was obtained from 2-mL clot blood samples.

Serology. The serological diagnosis was made using antigens of L. infantum prepared from a reference human strain (strain MCAN/IR/96/LON-49). IFA titer was determined from a serial dilution of 1:20 to 1:640 for each serum sample, according to WHO/LEISH/96.40 protocol. Human antiglobulin (Binding Site Co., Birmingham, UK) at a dilution of 1:300 was used. Control serum (titer 1:1,024) was collected from a patient with kala-azar.

DNA isolation. Peripheral blood (PB) was prepared for PCR amplification by the method described previously.11 Five hundred microliters of PB was incubated for 24 hours at 56°C in 2 volumes of proteinase K lysis buffer (0.5% Tween 20, 0.5% Nonidet P-40, 10 mmol/L NaOH, 10 mmol/L Tris [pH 7.2], 320 µg of proteinase K per milliliter) and boiled for 10 minutes. A simplified phenol-chloroform extraction was performed with 700 µL of this lysate, followed by ethanol precipitation and resuspension in 10 µL of sterile distilled water.

PCR-ELISA. To detect L. infantum kDNA, all samples were tested by PCR-ELISA.11 Amplification reactions for direct labeling of PCR product with digoxigenin was carried out using DIG-Labeling Kit (Roche Diagnostic, Mannheim, Germany). To detect PCR product by ELISA, we used a 5'-labeled internal oligonucleotide probe (18 bases) with a PCR-ELISA Kit (DIG Detection; Roche Diagnostic). Absorbance values ≥ 1 were considered positive. The absorbance values obtained for human DNA were always < 0.1. To check the quality of the results, a second DNA preparation and amplification, independent of the first, was done in 28 randomly selected samples.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Leishmania kDNA in PB. L. infantum kDNA was detected by PCR-ELISA in 95 (24.5%) of 388 samples. The percentage of PCR-positive samples did not show any significant differences in all age groups or regions. All PCR-positive individuals were IFAT positive. The data showed that the percentage of PCR-positive results increased with higher titer of antibody in serums (Table 1Go).


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TABLE 1
IFAT and PCR-ELISA of asymptomatic individuals in endemic areas, southern Iran, by different serum antibody titers
 
Serologic study. Anti-Leishmania antibodies were detected in 212 of 388 (54.6%) asymptomatic individuals using the IFAT method. Of 212 IFAT-positive individuals, 144 (67.9%) had low antibody titer of 1:20, 57 (26.9%) of 1:40, and 11 (5.2%) had titer of 1:80 (Table 2Go). Individuals 1–5 years of age had the highest seropositivity (69.8%; Table 2Go). kDNA was not detected in 117 (55.2%) seropositive individuals. It is worth mentioning that 86 (90.5%) of 95 PCR-positive samples had a low titer of IFA 1:20–1:40.


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TABLE 2
PCR-ELISA, IFAT, and LST of asymptomatic individuals in endemic areas, southern Iran, by different age groups
 
Leishmanin skin test. The LST was positive in 132 (34%) of individuals, and only 40 (30.3%) of the subjects were PCR positive. However, of 256 LST-negative subjects, 55 (21.4%) were PCR positive. LST positivity (45.6%) was highest among individuals 16–35 years of age (Table 2Go).


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Clinical presentation of leishmaniasis is dependent on immune status, species, and strain of parasite.12 The majority of infected cases are asymptomatic and do not progress to VL.13,14 The evaluation of infection in epidemiologic studies is traditionally carried out using serologic methods and LST.6 In asymptomatic individuals, because of poor humoral immunity response, sensitivity of serologic methods is low.15,16 The lack of sensitivity of LST as a valuable test to determination of prevalence is reported.6,17 PCR methods have been shown to be highly sensitive and specific in diagnosis of active VL, but their sensitivity and specificity in asymptomatic individuals, because of the lack of a gold standard, has not been determined.4–6,18

In this study, a high rate of asymptomatic infections (24.5%) was confirmed through detection of L. infantum DNA in the PB of healthy subjects in endemic areas. Previous studies performed in Spain and France are consistent with the results of this study.4,6 The results of PCR assays have been in a wide range of 7.4–50.8% in asymptomatic subjects in endemic areas, which could be explained by varying selected populations in studies and using PCR methods with different levels of sensitivity.3,5,19 According to numerous reports, it is assumed that specificity of PCR targeting mini-circle kDNA is 100%.7–11 It is unlikely that PCR-ELISA results are false positive, because we used primers and a probe that have been shown to be highly specific for L. infantum, and human DNA was used as a negative control.11,19

The high kDNA-PCR positivity indicates that asymptomatic infected subjects in endemic areas could act as a potential reservoir, and those who developed secondary immunodeficiency disorders such as HIV infection are vulnerable to reactivation of a latent infection.3,6,20–22

Meanwhile, it is noteworthy that positive kDNA-PCR in endemic area can not serve as a reliable test for the diagnosis of symptomatic kala-azar. In this situation, quantitative real-time PCR could determine the threshold between asymptomatic carriage and disease in endemic areas.20

In this study, 132 (34%) of LSTs were positive as previously reported, ranging from 9.7% to 57.8%.23–25 Our data showed no association between the results of kDNA PCR-ELISA and those of LST. A study performed on asymptomatic blood donors living in an endemic area in Spain is in agreement with these results and showed the results of nested PCR and LST as independent.6 Positive LST and PCR-ELISA have been detected in 40 individuals (30.3%), which means a cell-mediated immunity could not predict the parasite eradication. Ninety-two subjects (69.7%) had positive LST and negative PCR-ELISA, which may be related to the low density of Leishmania in PB and probable episodic parasitemia in asymptomatic infected individuals or may be caused by the detection of immunologic memory, in the absence of parasite, by LST.3,6 There were also 55 individuals (21.4%) with LST-negative results who were PCR positive, which could be caused by anergy or ignorance.26 Considering these findings, LST alone could not be used as a reliable index of prevalence rate of infection in endemic area and needs to be accompanied by kDNA PCR-ELISA.

In seroepidemiologic studies, the IFAT cut-off level to detect asymptomatic infected subjects has not yet been defined. In this study, a high proportion (54.4%) of healthy individuals had IFAT ≥ 1:20, but only 11 (2.8%) had titer of 1:80. In other endemic Mediterranean areas, the values ≥ 1:80 were positive in only 0.5–9.2% of asymptomatic humans, and in a study in Brazil, none of the asymptomatic subjects were positive for IFAT ≥ 1:80.18,27,28 L. infantum kDNA was detected only in individuals who were antibody positive, and an increase in serum antibody titer was associated with increased rate of PCR positivity (Table 1Go). These findings are in agreement with the results of the study carried out in southern Spain.4 In this study, the majority (90.5%) of PCR-positive samples had a titer of IFA 1:20–1:40, so the low titer of IFAT could be considered significant in seroepidemiologic studies, and in PCR-based epidemiologic studies, a positive IFAT titer (≥ 1:20) may be initially used for screening the subjects.

However, in 117 (55%) seropositive individuals with IFAT ≥ 1:20, kDNA was not detected. The negative kDNA PCR-ELISA in individuals with positive IFAT antibody may be caused by the persistent parasites in the reticoendothelial system and subsequent episodic release of parasites in the blood or a cross-reaction with other diseases such as tuberculosis, toxoplasmosis, brucellosis, and typhoid fever.1,3,29

Individuals 1–5 years of age had the highest seropositivity (69.8%), whereas LST positivity (45.6%) was highest among individuals belonging to the age group 16–35 years (Table 2Go). It seems that during earlier exposures that usually occur in subjects < 5 years in an endemic area, the immune response to L. infantum is a mixed humeral and cellular response. In asymptomatic subjects, the persistence of a parasite load could be a mechanism that permits the constant stimulation of cell-mediated immunity, as evidenced by the presence of positive LST. This can account for decreasing seropositivity and increasing LST with advancing age in asymptomatic individuals.

In conclusion, the high rate of kDNA-positive samples could show that humans may play a role in transmission in endemic areas. LST alone could not be used as a reliable index of prevalence rate of infection and needs to be accompanied by kDNA PCR-ELISA. In endemic areas, kDNA PCR-ELISA is not a confirmatory test for the diagnosis of active kala-azar.

Received February 18, 2008. Accepted for publication August 8, 2008.

Acknowledgments: The authors thank H. Khajehei, PhD, for valuable assistance and cooperation in copyediting the manuscript.

* Address correspondence to Abdolvahab Alborzi, Professor Alborzi Clinical Microbiology Research Center, Nemazi Hospital, Shiraz, Iran. E-mail: alborziiraj2004{at}yahoo.com Back

Authors’ addresses: Abdolvahab Alborzi, Bahman Pourabbas, Fra-had Shahian, Jalal Mardaneh, Gholam R. Pouladfar, and Mazyar Ziyaeyan, Professor Alborzi Clinical Microbiology Research Center, Nemazi Hospital, Shiraz, Iran, Tel: 98-711-6470205, Fax: 98-711-6287071, E-mails: alborziiraj2004{at}yahoo.com, bpourabbas{at}yahoo.com, farhadwww{at}yahoo.com, jalalmardaneh{at}yahoo.com, pooladfar{at}yahoo.com, and ziyaeyan{at}gmail.com.


REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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