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Serological survey of Leishmaniainfection in blood donors in Salvador, Northeastern Brazil

  • Kiyoshi F Fukutani1,
  • Virgínia Figueiredo2,
  • Fabiana S Celes1,
  • Juqueline R Cristal1,
  • Aldina Barral1, 3,
  • Manoel Barral-Netto1, 3 and
  • Camila I de Oliveira1, 3Email author
BMC Infectious Diseases201414:422

DOI: 10.1186/1471-2334-14-422

Received: 26 February 2014

Accepted: 24 July 2014

Published: 30 July 2014

Abstract

Background

Visceral Leishmaniasis is endemic to Brazil, where it is caused by Leishmania infantum (syn. Leishmania chagasi). Following parasite inoculation, individuals may experience asymptomatic infection, raising the possibility of parasite transmission through the transfusion of contaminated blood products. In the present work, we evaluated the prevalence of asymptomatic Leishmania infection among blood donors in Salvador, northeastern Brazil.

Methods

Peripheral blood was collected from 700 blood donors attending the Blood Bank of Bahia (HEMOBA/SESAB), from January to September 2010. We evaluated anti-Leishmania serology by ELISA, employing Soluble Leishmania Antigen (sensitivity 90% and specificity 95%). The presence of parasite DNA was determined by qPCR, targeting a single copy gene (G6PD), and by end-point PCR, targeting multiple targets, namely a segment located in the Leishmania rRNA locus (ITS) and the conserved region of kinetoplastid DNA (kDNA) minicircles.

Results

The blood-donor population was comprised of 74.5% of males with a mean age of 34 years. Anti-Leishmania serology by ELISA was positive in 5.4% (38/700) individuals. One individual was also positive for Chagas’ disease and another tested positive for Syphilis. Employing qPCR, parasite DNA was not found in any of 38 seropositive samples. However, by ITS PCR, 8/38 (21%) samples were positive and this positivity increased to 26/38 (68%) when we targeted kDNA amplification. Agreement between both techniques (ITS and kDNA PCR) was fair (kappa = 0.219).

Conclusions

These results indicate that asymptomatic infection is present among the blood donor population of Salvador, a finding that warrants a broader discussion regarding the need to implement specific screening strategies.

Keywords

Leishmania Blood supply Asymptomatic infection PCR ELISA

Background

Visceral leishmaniasis (VL) or kalazar is a serious public health problem worldwide, and approximately 500,000 new cases are reported each year {[1] #67}. Leishmania infantum is the causative agent of VL in Brazil, which accounts for 90% of cases in the Americas {[2] #257} and where 3392 VL cases were reported in 2012 {[3], #258}. In the last decades, VL is becoming more prevalent in urban centers of Brazil, especially in expanding cities with different patterns of economic and social development {[4] #259}. Patent VL is classically characterized by the presence of irregular fever, paleness, and splenomegaly and, in the absence of treatment; it is a progressive infection with fatal outcome {[3] #279}. However, a large number of infections remain asymptomatic, in which individuals are infected by Leishmania but display an apparent healthy condition (rev. in {[5] #260}). There are concerns that asymptomatic individuals remain carriers of the infection and, as such, may play an important role in the epidemiology of VL, especially in urban concentrations where the disease is present {[6] #261}.

Classically, transmission of Leishmania parasites occurs through sand fly bites, however, studies in France {[7] #248}, Italy {[8] #6}, Spain {[9] #12}, India {[10] #9}, Bangladesh {[11] #250} and Turkey {[12] #251} showed that asymptomatic individuals are found among blood donors, suggesting that transfusion of Leishmania-contaminated blood products might be a source of disease transmission in parallel to syringe sharing {[13] #68} and organ transplantation {[14] #7; [15] #8; [16] #19; [17] #41; [18] #246}. In northeastern Brazil, the prevalence of anti-Leishmania antibodies was 9% among blood donors, increased to 25% in a VL focus and was highest (37%) among poly-transfused hemodialysis patients {[19] #37}. In a subsequent study, upon evaluation of 21 sero-positive blood-donors, authors found that five individuals (23%) were positive to Leishmania by PCR whereas dot-blot hybridization increased this positivity to nine individuals (43%) {[20] #10}. In central western Brazil, the prevalence of anti-Leishmania antibodies among a population of blood donors was reported at 15.6% {[21] #262}, indicating the presence of asymptomatic individuals among blood donors in different regions of the country.

Herein, our goal was to determine the prevalence of anti-Leishmania antibodies among blood donors of Salvador, Bahia, situated in northeastern Brazil. Although Salvador city per se is not endemic for VL, the HEMOBA/SESAB central blood bank draws donors from all over Bahia state and, as such, consists of a primordial sample for large-scale investigation. Of note, Bahia accounted for ~8% of VL cases registered in Brazil in 2012 and in 2013 {[22], #258}. We investigated both the presence of anti-Leishmania antibodies and of parasite DNA. For the latter, we employed a quantitative Real Time PCR (qPCR) {[23] #24}, targeting a single copy gene, and two conventional (end-point) PCR assays targeting: i) the Internal Transcribed Spacers present in rRNA {[24] #252} and ii) the conserved region of minicircle kDNA {[25] #253}.

Methods

Ethical considerations

This study was approved by the research ethics committee of CPqGM-FIOCRUZ-BA (No. 215/2010). Individuals involved in this study were required to sign the Informed Consent Form.

Study design and population

The purpose of this study was to identify asymptomatic infection in blood donors attending the Hematology and Hemotherapy Foundation of Bahia State (HEMOBA/SESAB), situated in Salvador, BA, Brazil. The necessary sample size of approximately 220 individuals was estimated according to the following parameter: prevalence of an asymptomatic infection rate of 9%, based on a study conducted in Natal {[19] #37}, a VL endemic area {[26] #254}. Blood was obtained from 700 donors attending HEMOBA/SESAB and blood donations were screened over a period of nine months (January to September 2010). Donors resided in Salvador and other municipalities from BA.

Sample collection

Whole blood was collected in quadruple blood packs (Fresenius/KAB), using the top-and-bottom system. Aliquots of 5 ml of peripheral blood were obtained following blood collection and were placed into Vacutainer® tubes (Becton Dickinson) containing Heparin as anti-coagulant. Serum was separated by centrifugation and stored at -20°C for subsequent serologic studies. Aliquots of 200 μl of blood were used for DNA extraction.

Soluble Leishmaniaantigen

SLA was prepared from L. infantum MCAN/BR/00/BA262) and from L. amazonensis (MHOM/BR/1985/BA32) promastigotes maintained in Schneider (LGC Scientific) medium (Sigma) supplemented with 10% inactivated fetal bovine serum, 100 U/ml penicillin and 100 ug/ml streptomycin (Life Technologies). The parasites were initially submitted to 10 alternating cycles of freezing in liquid nitrogen and thawing in a water bath and were then centrifuged at 1600 × g, 4°C for 15 min. The supernatant containing SLA was collected and protein content was quantified using the Micro BCA TM Protein Reagent Kit assay (Pierce).

ELISA to detect anti-Leishmaniaantibodies

Anti-Leishmania serology was performed by ELISA, as described {[27] #20}. Briefly, 96-well plates (Linbro/Titertek) were coated with 10 μg/mL of antigen (L. infantum or L. amazonensis SLA) and were incubated overnight at 4°C. After three washes with PBS/0.5% Tween, plates were blocked for 1 hour at 37°C with PBS/Tween 0.5% plus 1% fetal bovine serum (FBS) (Life Technologies). Plasma samples, diluted 1:100 in PBS/Tween 0.5% plus 1% FBS were added in duplicate and plates were incubated for 2 hours at room temperature. After three rounds of washing, wells were incubated with anti-human IgG conjugated to alkaline phosphatase (SIGMA) at a 1:2500 dilution in PBS/Tween 0.5% plus 1% FBS for 1 hour at room temperature. Plates were then washed and were further incubated for 30 minutes with a chromogenic solution of p-nitrophenyl phosphate (SIGMA) in sodium carbonate buffer pH 9.6 containing 1 mg/mL of MgCl2. In all experiments, the values obtained were subtracted from those obtained in the background. ELISA experiments were repeated twice yielding similar results. The ELISA cut-off value for L. infantum and L. amazonensis SLA was established using ROC curves (please see Statistical analysis below). In this case, we employed a panel of positive sera (n = 20), obtained from VL patients [defined by clinical and laboratory signs {[28] #280} and a positive bone marrow aspirate]. Control sera (n = 20) were obtained from negative individuals, living in areas free of L. infantum. These sera were randomly selected from a serum bank (LIP-CPqGM-FIOCRUZ).

DNA extraction

DNA was extracted from 200 μL of whole blood using Illustra Genomic Prep Blood Mini Spin Kit™ (GE Healthcare), following the manufacturer’s instructions. DNA was eluted into 200 μL of Elution buffer and stored at -20°C. Purity of genetic material was assessed by absorbance at 260/280 nm.

Real-Time PCR

Detection of Leishmania DNA by Real Time PCR was performed as described by {[23] #24}. Briefly, real time PCR was conducted in an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems) employing SYBR-Green® PCR Master Mix (Invitrogen), 50 ng DNA and 200 nM of each forward and reverse primers. Thermal cycle conditions consisted of a denaturation step at 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Oligonucleotides (Fwd G6PD 5′-CCCGAGGGCAGCACTTG-3′ and Rev G6PD 3′-CCACCGGTCGTTGTTGATG-5′) were designed using PrimerExpress3.0 (Applied Biosystems) and were based on the L. infantum G6PD gene (glucose-6-phosphate dehydrogenase) (GeneBank: DQ212794.1). For parasite quantification, a standard curve was constructed based on DNA extracted from serial dilutions of L. infantum, ranging from 106 to 101 parasites. Analysis and acquisition of data were performed with SDS software (Applied Biosystems).

Conventional PCR

End-point PCR targeting the 120-bp conserved region of the Leishmania kDNA minicircles {[25] #253} was performed as described {[29] #264}, employing primers 5′-GGG (G/T)AGGGGCGTTCT(G/C)CGAA-3′ and 5′-(G/C)(G/C)(G/C)(A/T)CTAT(A/T)TTACACCAACCC C-3′. The second end-point PCR assay targeted amplification of the segment containing ITS 1, 2 and the 5.8S region, within the rRNA gene locus {[24] #252}. Primers employed were IR1 (5′-GCTGTAGGTGAACCTGCAGCAGCTGGATCATT-3′) and IR2 (5′-GCGGGTAGTCCTGCCAAACACTCAGGTCTG-3′). Reactions were performed in a final volume of 20 μl, with 50 ng of DNA, 0.2 μM of each oligonucleotide and PCR supermix (Invitrogen), following manufacturer’s instructions. PCR conditions were as follows: denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 60 sec, 55°C for 60 sec and 94°C for 120 sec with a final extension of 72°C for 10 min. The amplification reactions were analyzed by agarose gel electrophoresis, followed by silver or ethidium bromide staining. DNA from the reference L. infantum strain was used as positive control for PCR reactions.

Statistical analysis

Statistical analysis was performed using Prism v.5.0 (Graph Pad Software). ROC (Receiver Operating Characteristic) curves were calculated based on ELISA assays performed with SLA obtained from L. amazonensis and L. infantum. The cut-off, as determined by high sensitivity and specificity, from the highest probability of discrimination established by the curve, was therefore determined. The performance of each SLA was established by the parameters obtained from ROC curves values (AUC, p values and likelihood ratio). Non-parametric Mann–Whitney test was used to compare the frequency of seropositive individuals with a positive PCR result or a negative PCR result. The p value was considered significant when < 0.05. Agreement between PCR assays targeting Leishmania ITS and kDNA were determined by the kappa index.

Results

Anti-L. infantumserology in blood donors recruited at HEMOBA/SESAB

Initially, we determined the sensitivity and specificity of the Soluble Leishmania Antigen (SLA) preparations [obtained from L. infantum or from L. amazonensis (the latter an etiological agent of Cutaneous Leishmaniasis)] when employed as the capture antigen in ELISA assays. With the results obtained, we constructed two ROC curves (Figure 1). L. infantum SLA performed better (AUC: 0.99, p < 0.0001, Likelihood ratio: 18.0) (Figure 1) when compared to L. amazonensis SLA (AUC 0.87; p < 0.0001 and Likelihood Ratio 15.0). Therefore, SLA from L. infantum presented higher sensitivity and specificity when compared to SLA from L. amazonensis and was selected for subsequent experiments. The cut off value, based on higher sensitivity and specificity of L. infantum SLA, was established as 0.01685.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2334-14-422/MediaObjects/12879_2014_Article_3718_Fig1_HTML.jpg
Figure 1

ROC curve of antibody levels predict the positivity thresholds against SLA. (A) ROC curves were constructed using data obtained in ELISA performed with SLA from L. infantum (L. i) or from L. amazonensis (L.a) and sera from confirmed VL patients (n = 20) and from control subjects from a non-endemic (n = 20) area. The red line represents the area under the curve for L.ic and the blue line represents the area under the curve for L.a. (B) Detailed information obtained for each ROC curve (cut-off values chosen, area under the curve, the values of p, and the sensitivity and specificity with a confidence interval of 95% and the likelihood ratio) is shown on the right.

Cross-reaction among Leishmaniaand other blood borne pathogens

Next, we investigated the presence of anti-Leishmania antibodies in the sera from 700 blood donors (74.5% male) attending HEMOBA/SESAB. The median age was 42 years: 31.7% individuals had between 19 and 28 years, 32.8% between 28 and 38 years, and 34% were older than 38 years of age. The reactivity of blood donors’ sera to L. infantum SLA was significantly higher compared to that obtained with negative controls (Figure 2). As expected, the reactivity of sera from confirmed VL patients was higher (p < 0.0001) compared to both blood donors and negative controls (Figure 2). Among the blood donors, we identified 38 individuals (5.4%) (81.5% male) that reacted above the established cut-off value (0.01685) and were thus considered seropositive against L. infantum. These 38 samples were also tested for cross-reactivity to other agents, following the routine blood screening investigation performed at HEMOBA/SESAB. Among the 38 Leishmania seropositive samples, 1/38 (0.02%) reacted positive for Chagas’ disease whereas another sample (1/38) reacted positively for Syphilis. Of note, among the 38 blood donors that were seropositive for Leishmania, 26/38 (68%) currently live in Salvador whereas 8/38 (21%) live in adjacent municipalities (address information was missing from four samples).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2334-14-422/MediaObjects/12879_2014_Article_3718_Fig2_HTML.jpg
Figure 2

Detection of Leishmania antibodies in blood donors of Salvador. Samples from blood donors (n = 700) were tested for reactivity against L. infantum SLA by ELISA. Positive controls (n = 55) consisted of sera from individuals diagnosed with clinical VL and negative controls samples (n = 38) were obtained from volunteers living in a non-endemic area. All samples were tested in duplicate. ***p < 0.001.

Detection of LeishmaniaDNA in blood donors’ samples

We then investigated the parasite load in the 38 (5.4%) Leishmania seropositive samples (Figure 2). To do so, DNA extracted from the 38 blood samples was submitted to quantitative PCR (qPCR) targeting the single copy G6PD gene {[30] #243}. A standard curve (threshold cycle versus logarithm of the amplicon copy number) was constructed using DNA extracted from L. infantum, ranging 106 to 101 parasites (Figure 3). The standard curve showed a good linear correlation (R2 = 0.971) when plotted against the DNA equivalent to the number of parasites and the efficiency of the assay was 97%. We did not detect any level of interference due to the presence of human DNA as a similar efficiency of amplification was obtained when standard curves were generated from parasite DNA spiked with human DNA or from DNA extracted from a mixture of parasites plus human DNA (data not shown). Using the G6PD qPCR assay, we were not able to detect Leishmania DNA in any of the 38 blood donors that were seropositive by ELISA (Table 1). We then investigated whether end-point PCR assays targeting multiple copy genes would improve detection of parasite DNA. Targeting the region located between the small and the large subunit of the Leishmania rRNA gene array, including the Internal Transcribed Spacers (ITS) and the 5.8S rRNA {[24] #252}, we detected positive amplification in 8/38 (21.0%) samples (Table 1). In another assay, we amplified the conserved region of minicircles, present in 10,000-20,000 copies, located within the Leishmania kinetoplast {[25] #253}. This assay was positive in 26/38 (68.4%) samples (Table 1), raising the detection level by 47,4% when compared to ITS PCR (Table 1). The concordance between kDNA and ITS PCR was fair (kappa = 0.219) (Table 2). Lastly, positivity in either PCR assays, ITS or kDNA amplification, was dissociated from serology OD levels, determined by ELISA (Figure 4A and B, respectively).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2334-14-422/MediaObjects/12879_2014_Article_3718_Fig3_HTML.jpg
Figure 3

Standard curve obtained with the G6PD L. infantum qPCR test. The standard curve was generated by plotting the Ct against the input target quantity (in this case, serial dilutions of Leishmania DNA respresenting 106 to 101 parasites). Ct represents the fractional cycle number reflecting a positive PCR result differentiated from the background noise. Slope -3.206, PCR efficacy 105%, R2 = 0.971.

Table 1

Detection of Leishmania DNA in blood donors (n = 38) from Salvador, seropositive for Leishmania

PCR

G6PD {[30] #243}

ITS {[24] #252}

kDNA {[25] #253}

Positive, n (%)

0

8 (21.1)

26 (68.4)

Negative, n (%)

38 (100)

30 (78.9)

12 (31.6)

Total

38

38

38

Table 2

Agreement between kDNA and ITS PCR results in blood donors (n = 38) with anti- Leishmania antibodies

 

kDNA

 

ITS

Positive (%)

Negative (%)

Total

Positive, n (%)

8 (21)

0

8

Negative, n (%)

18 (47)

12 (31)

30

Total

26

12

38

Kappa = 0.219.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2334-14-422/MediaObjects/12879_2014_Article_3718_Fig4_HTML.jpg
Figure 4

Comparison of ELISA OD values obtained for blood donors with distinct PCR results. Samples that were either negative or positive by ITS amplification (A) or kDNA amplification (B) were plotted against the respective OD values obtained by ELISA. Data are shown as the median OD obtained for each sample. Numbers above indicate the number of samples in each category (PCR negative or PCR positive).

Discussion

Asymptomatic Leishmania infantum infection can be detected initially with antibodies {[31] #275}; antibody responses decline with time in parallel to the development of a cell mediated immune response {[32] #282}. Therefore, serologic responses to Leishmania indicate acute infection, in the presence of symptoms or not. Reports of Leishmania transmission through the use of contaminated blood products {[14] #7; [10] #9} have raised the question whether asymptomatic infection among blood donors poses a problem for the blood supply, especially in regions where VL is endemic. Presently, we investigated the anti-Leishmania serologic response among blood donors from Salvador, northeastern Brazil. Additionally, we employed three PCR-based assays to determine the presence of Leishmania DNA in samples with positive serology.

Among the 700 blood donors recruited at HEMOBA/SESAB, the major blood donation center in Salvador, we detected 38 individuals (5.4%) with a positive serology to Leishmania. This finding is similar to that reported in Montes Claros (5.5%) {[33] #265} but lower than that observed in Natal (9%) {[20] #10} and in Campo Grande (15.6%) {[21] #262}, both of which are endemic regions for VL. The lower prevalence detected in our study may be due to the fact that Salvador is not a VL endemic area per se, differently from the other three municipalities. Additionally, differences in the sensitivity of the tests employed herein (SLA-based ELISA) versus those used in Natal (FML-ELISA) {[20] #10}, Montes Claros and Campo Grande (IFAT) {[33] #265; [21] #262} may contribute for the differing results. In our study, an ELISA assay employing L. amazonensis SLA was less sensitive when compared to L. infantum SLA (Figure 1), suggesting that the choice of antigen impacts on the results obtained. Among the 38 seropositive individuals detected herein, only one tested positive for Chagas disease under the routine screening performed at HEMOBA. We suggest that, in this current setting, routine screening for Chagas’ disease is not excluding Leishmania infection.

Molecular techniques have been successfully used to detect asymptomatic Leishmania infection {[34] #28; [26] #254; [35] #40; [36] #272} {[37] #273} and for the diagnosis and follow-up of VL patients {[38] #266; [39] #267}. Herein, we employed PCR to detect parasite DNA in the 38 individuals presenting anti-Leishmania antibodies. A quantitative assay, based on amplification of a single-copy target (G6PD) {[30] #243}, failed to amplify parasite DNA within the range established by the standard curve. This assay, however, was successful at identifying and estimating the number of parasites in biopsy samples from Cutaneous Leishmaniasis patients {[30] #243}. One possible reason can be the presence of very low levels of circulating parasites, hampering amplification of a single-copy gene. However, in a study conducted in Natal state, Brazil, positivity by qPCR (17%) was lower compared to positivity by ELISA (24.6%), even when a repetitive region was used as the amplification target {[26] #254}. In Minas Gerais state, Brazil, authors detected 56.5 parasites/ml of blood from seropositive individuals and this rate decreased to 7.8 parasites/ml after a one-year follow up, as measured by qPCR also targeting a repetitive region {[40] #270}. Indeed, a number of studies have reported differences in positivity by serological and molecular tests to detect Leishmania {[41] #31; [20] #10; [42] #255, [43] #13 [11] #250}. Herein, when we employed conventional end-point PCR targeting the ITSs within the rRNA gene array {[24] #252} or the conserved region within kDNA minicircles {[25] #253}, we were able to amplify Leishmania DNA in 8/38 (21%) and 28/38 (68%) seropositive samples, respectively, and agreement between both techniques was fair (kappa = 0.219). In a study with HIV/AIDS patients, prevalence of Leishmania based on ITS PCR was 1.8% {[44] #268} whereas by kDNA PCR, it increased to 11.8%, in France {[41] #31}, to 24% in Spain {[42] #255} and to 36.4% in Italy {[43] #14}. These results are in accordance with our observation that the higher the number of repetitions of a given amplification target, such as the ITSs or kDNA, the higher the sensitivity of the assay {[45] #278}. In our hands, kDNA amplification also proved a useful tool for the diagnosis of Cutaneous {[29] #264} and Mucocutaneous Leishmaniasis {[46] #281}. The fact that either ITS or kDNA amplification was negative in 12/38 samples (31.6%) with a positive Leishmania serology could again be explained by the presence of very low parasite numbers and, hence, few targets were available for PCR amplification. Another hypothesis is the presence of parasite remnants within phagocytic cells, rendering a positive amplification of Leishmania DNA by PCR.

In the current setting, blood screening for the presence of VL would decrease blood supply by 5%, negatively impacting on the blood supply provided by HEMOBA/SESAB. Hamsters transfused with blood from an experimentally infected animal developed disease as shown by presence of ascites, cachexia and death {[47] #44}. Foxhounds that received packed red blood cell transfusions from seropositive donors also tested positive for Leishmania antibodies {[48] #43}, indicating transmission of Leishmania to recipient dogs by blood transfusion. In a study conducted in Bangladesh, 1.195 blood donors were screened, antibodies were found in three individuals but none developed VL during a 6 month follow-up period {[11] #250}, suggesting that the number of circulating parasites is below the threshold necessary to initiate symptoms.

Conclusions

Given that positive serology and/or presence of parasite DNA does not guarantee transfusional risk, the importance of our results lies in the recognition of the presence of cryptic Leishmania infection among blood donors from Salvador. Leishmania testing is not mandatory in blood banks in Brazil but knowledge of positive serology would impose blood elimination. Since VL is expanding and control measures are not efficient, a broader discussion is needed regarding the need to implement specific screening strategies and, possibly, the risk of Leishmania transmission by blood transfusion.

Abbreviations

ELISA: 

Enzyme-linked Immunosorbent Assay

IFAT: 

Immunofluorescence Antibody Test

ITS: 

Internal Transcribed Spacer

kDNA: 

Kinetoplastid DNA

PCR: 

Polymerase chain reaction

SLA: 

Soluble Leishmania Antigen

VL: 

Visceral Leishmaniasis.

Declarations

Acknowledgments

This work was supported by grants from FIOCRUZ and CNPq. KFF was a recipient of a CAPES fellowship. FSC was a recipient of a PIBIC/FIOCRUZ fellowship. AB, MBN and CIO are senior investigators from CNPq.

Authors’ Affiliations

(1)
Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz (FIOCRUZ)
(2)
Fundação de Hematologia e Hemoterapia da Bahia (HEMOBA/SESAB)
(3)
Instituto de Investigação em Imunologia

References

  1. Leishmaniasis. http://www.who.int/leishmaniasis,
  2. Romero GA, Boelaert M: Control of visceral leishmaniasis in latin america-a systematic review. PLoS Negl Trop Dis. 2010, 4 (1): e584-View ArticlePubMedPubMed CentralGoogle Scholar
  3. Lainson R, Shaw JJ: Epidemiology and ecology of leishmaniasis in latin-America. Nature. 1978, 273 (5664): 595-600.View ArticlePubMedGoogle Scholar
  4. Harhay MO, Olliaro PL, Costa DL, Costa CH: Urban parasitology: visceral leishmaniasis in Brazil. Trends Parasitol. 2011, 27 (9): 403-409.View ArticlePubMedGoogle Scholar
  5. Michel G, Pomares C, Ferrua B, Marty P: Importance of worldwide asymptomatic carriers of leishmania infantum (L. chagasi) in human. Acta Trop. 2011, 119 (2–3): 69-75.View ArticlePubMedGoogle Scholar
  6. (WHO) WHO: Leishmaniasis. Fact sheet. 2013, n.375: http://www.who.int/mediacentre/factsheets/fs375/en/,Google Scholar
  7. Kubar J, Quaranta JF, Marty P, Lelievre A, Le Fichoux Y, Aufeuvre JP: Transmission of L. infantum by blood donors. Nat Med. 1997, 3 (4): 368-View ArticlePubMedGoogle Scholar
  8. Cardo LJ: Serological screening for Leishmania infantum in asymptomatic blood donors living in an endemic area (Sicily, Italy). Transfus Apher Sci. 2006, 34 (2): 233-234.View ArticlePubMedGoogle Scholar
  9. Riera C, Fisa R, Lopez-Chejade P, Serra T, Girona E, Jimenez M, Muncunill J, Sedeno M, Mascaro M, Udina M, Gállego M, Carrió J, Forteza A, Portús M: Asymptomatic infection by Leishmania infantum in blood donors from the Balearic Islands (Spain). Transfusion. 2008, 48 (7): 1383-1389.View ArticlePubMedGoogle Scholar
  10. Dey A, Singh S: Transfusion transmitted leishmaniasis: a case report and review of literature. Indian J Med Microbiol. 2006, 24 (3): 165-170.PubMedGoogle Scholar
  11. Huda MM, Rudra S, Ghosh D, Bhaskar KR, Chowdhury R, Dash AP, Bhattacharya SK, Haque R, Mondal D: Low prevalence of Leishmania donovani infection among the blood donors in kala-azar endemic areas of Bangladesh. BMC Infect Dis. 2013, 13: 62-View ArticlePubMedPubMed CentralGoogle Scholar
  12. Ates SC, Bagirova M, Allahverdiyev AM, Baydar SY, Koc RC, Elcicek S, Abamor ES, Oztel ON: Detection of antileishmanial antibodies in blood sampled from blood bank donors in Istanbul. Future Microbiol. 2012, 7 (6): 773-779.View ArticlePubMedGoogle Scholar
  13. Cruz I, Morales MA, Noguer I, Rodriguez A, Alvar J: Leishmania in discarded syringes from intravenous drug users. Lancet. 2002, 359 (9312): 1124-1125.View ArticlePubMedGoogle Scholar
  14. Cohen C, Corazza F, De Mol P, Brasseur D: Leishmaniasis acquired in Belgium. Lancet. 1991, 338 (8759): 128-View ArticlePubMedGoogle Scholar
  15. Cummins D, Amin S, Halil O, Chiodini PL, Hewitt PE, Radley-Smith R: Visceral leishmaniasis after cardiac surgery. Arc Dis Child. 1995, 72 (3): 235-236.View ArticleGoogle Scholar
  16. Basset D, Faraut F, Marty P, Dereure J, Rosenthal E, Mary C, Pratlong F, Lachaud L, Bastien P, Dedet JP: Visceral leishmaniasis in organ transplant recipients: 11 new cases and a review of the literature. Microbes Infect. 2005, 7 (13): 1370-1375.View ArticlePubMedGoogle Scholar
  17. Mpaka MA, Daniil Z, Kyriakou DS, Zakynthinos E: Septic shock due to visceral leishmaniasis, probably transmitted from blood transfusion. J Infect Dev Ctries. 2009, 3 (6): 479-483.PubMedGoogle Scholar
  18. Oliveira RA, Silva LS, Carvalho VP, Coutinho AF, Pinheiro FG, Lima CG, Leandro JE, Silva GB, Daher EF: Visceral leishmaniasis after renal transplantation: report of 4 cases in northeastern Brazil. Transpl Infect Dis. 2008, 10 (5): 364-368.View ArticlePubMedGoogle Scholar
  19. Luz KG, da Silva VO, Gomes EM, Machado FC, Araujo MA, Fonseca HE, Freire TC, D’Almeida JB, Palatnik M, Palatnik-de Sousa CB: Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients. Am J Trop Med Hyg. 1997, 57 (2): 168-171.PubMedGoogle Scholar
  20. Otero AC, da Silva VO, Luz KG, Palatnik M, Pirmez C, Fernandes O, de Sousa CB P: Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors. Am J Trop Med Hyg. 2000, 62 (1): 128-131.PubMedGoogle Scholar
  21. França Ade O, de Castro VL, Lima MS, Pontes ER, Dorval ME: Anti-leishmania antibodies in blood donors from the Midwest region of Brazil. Transfus Apher Sci. 2013, 49: 627-630.View ArticlePubMedGoogle Scholar
  22. DATASUS: Leishmaniose viscceral - casos confirmados notificados no sistema de informação de agravos de notificação - sinan net. http://dtr2004.saude.gov.br/sinanweb/tabnet/dh?sinannet/leishvi/bases/leishvbrnet.def,
  23. Castilho TM, Shaw JJ, Floeter-Winter LM: New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol. 2003, 41 (2): 540-546.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Cupolillo E, Grimaldi Junior G, Momen H, Beverley SM: Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol. 1995, 73 (1–2): 145-155.View ArticlePubMedGoogle Scholar
  25. Rodgers MR, Popper SJ, Wirth DF: Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitolo. 1990, 71 (3): 267-275.View ArticleGoogle Scholar
  26. Lima ID, Queiroz JW, Lacerda HG, Queiroz PV, Pontes NN, Barbosa JD, Martins DR, Weirather JL, Pearson RD, Wilson ME, Jeronimo SMB: Leishmania infantum chagasi in northeastern Brazil: asymptomatic infection at the urban perimeter. Am J Trop Med Hyg. 2012, 86 (1): 99-107.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Boaventura VS, Cafe V, Costa J, Oliveira F, Bafica A, Rosato A, de Freitas LA, Brodskyn C, Barral-Netto M, Barral A: Concomitant early mucosal and cutaneous leishmaniasis in Brazil. Am J Trop Med Hyg. 2006, 75 (2): 267-269.PubMedGoogle Scholar
  28. Saúde MD: Leishmaniose visceral: recomendações clínicas para redução da letalidade. Série A Normas e Manuais Técnicos. Edited by: Epidemiológica. BMdSSdVeSDdV. 2011, Brasília: Ministério da Saúde, 78-Google Scholar
  29. de Oliveira CI, Bafica A, Oliveira F, Favali CB, Correa T, Freitas LA, Nascimento E, Costa JM, Barral A: Clinical utility of polymerase chain reaction-based detection of Leishmania in the diagnosis of American cutaneous leishmaniasis. Clin Infect Dis. 2003, 37 (11): e149-e153.View ArticlePubMedGoogle Scholar
  30. Castilho TM, Camargo LM, McMahon-Pratt D, Shaw JJ, Floeter-Winter LM: A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase. Am J Trop Med Hyg. 2008, 78 (1): 122-132.PubMedGoogle Scholar
  31. Evans TG, Teixeira MJ, Sousa Ade Q, Pearson RD: Short report: extended follow-up of the natural history of persons infected with Leishmania chagasi. Am J Trop Med Hyg. 1995, 53 (4): 360-361.PubMedGoogle Scholar
  32. Badaro R, Jones TC, Carvalho EM, Sampaio D, Reed SG, Barral A, Teixeira R, Johnson WD: New perspectives on a subclinical form of visceral leishmaniasis. J Infect Dis. 1986, 154 (6): 1003-1011.View ArticlePubMedGoogle Scholar
  33. Urias EVR, Carvalho SFG, Preval Oliveira CL, Carvalho MLM, Teles LF, Rodrigues MC, Maia CN: Leishmania chagasieishmania species on the basis of glucose-6-phosphate de. Rev Bras Hematol Hemoter. 2009, 31: 348-354.View ArticleGoogle Scholar
  34. Costa CH, Stewart JM, Gomes RB, Garcez LM, Ramos PK, Bozza M, Satoskar A, Dissanayake S, Santos RS, Silva MR, Shaw JJ, David JR, Maguire JH: Asymptomatic human carriers of Leishmania chagasi. Am J Trop Med Hyg. 2002, 66 (4): 334-337.PubMedGoogle Scholar
  35. Moreno EC, Melo MN, Lambertucci JR, Serufo JC, Andrade AS, Antunes CM, Genaro O, Carneiro M: Diagnosing human asymptomatic visceral leishmaniasis in an urban area of the State of Minas Gerais, using serological and molecular biology techniques. Rev Soc Bras Med Trop. 2006, 39 (5): 421-427.View ArticlePubMedGoogle Scholar
  36. Orsini M, Canela JR, Disch J, Maciel F, Greco D, Toledo A, Rabello A: High frequency of asymptomatic Leishmania spp. infection among HIV-infected patients living in endemic areas for visceral leishmaniasis in Brazil. Trans R Soc Trop Med Hyg. 2012, 106 (5): 283-288.View ArticlePubMedGoogle Scholar
  37. Clemente WT, Rabello A, Faria LC, Peruhype-Magalhaes V, Gomes LI, da Silva TA, Nunes RV, Iodith JB, Protil KZ, Fernandes HR, Cortes JR, Lima SS, Lima AS, Romanelli RM: High prevalence of asymptomatic Leishmania spp. infection among liver transplant recipients and donors from an endemic area of Brazil. Am J Transplant. 2014, 14 (1): 96-101.View ArticlePubMedGoogle Scholar
  38. Lachaud L, Dereure J, Chabbert E, Reynes J, Mauboussin JM, Oziol E, Dedet JP, Bastien P: Optimized PCR using patient blood samples for diagnosis and follow-up of visceral Leishmaniasis, with special reference to AIDS patients. J Clin Microbiol. 2000, 38 (1): 236-240.PubMedPubMed CentralGoogle Scholar
  39. Mary C, Faraut F, Drogoul MP, Xeridat B, Schleinitz N, Cuisenier B, Dumon H: Reference values for Leishmania infantum parasitemia in different clinical presentations: quantitative polymerase chain reaction for therapeutic monitoring and patient follow-up. Am J Trop Med Hyg. 2006, 75 (5): 858-863.PubMedGoogle Scholar
  40. dos Santos Marques LH, Gomes LI, da Rocha IC, da Silva TA, Oliveira E, Morais MH, Rabello A, Carneiro M: Low parasite load estimated by qPCR in a cohort of children living in urban area endemic for visceral leishmaniasis in Brazil. PLoS Negl Trop Dis. 2012, 6 (12): e1955-View ArticlePubMedPubMed CentralGoogle Scholar
  41. le Fichoux Y, Quaranta JF, Aufeuvre JP, Lelievre A, Marty P, Suffia I, Rousseau D, Kubar J: Occurrence of Leishmania infantum parasitemia in asymptomatic blood donors living in an area of endemicity in southern France. J Clin Microbiol. 1999, 37 (6): 1953-1957.PubMedPubMed CentralGoogle Scholar
  42. Martin-Sanchez J, Pineda JA, Morillas-Marquez F, Garcia-Garcia JA, Acedo C, Macias J: Detection of Leishmania infantum kinetoplast DNA in peripheral blood from asymptomatic individuals at risk for parenterally transmitted infections: relationship between polymerase chain reaction results and other Leishmania infection markers. Am J Trop Med Hyg. 2004, 70 (5): 545-548.PubMedGoogle Scholar
  43. Scarlata F, Vitale F, Saporito L, Reale S, Vecchi VL, Giordano S, Infurnari L, Occhipinti F, Titone L: Asymptomatic Leishmania infantum/chagasi infection in blood donors of western Sicily. Tran R Soc Trop Med Hyg. 2008, 102 (4): 394-396.View ArticleGoogle Scholar
  44. Carranza-Tamayo CO, de Assis TS, Neri AT, Cupolillo E, Rabello A, Romero GA: Prevalence of Leishmania infection in adult HIV/AIDS patients treated in a tertiary-level care center in Brasilia, Federal District, Brazil. Tran R Soc Trop Med Hyg. 2009, 103 (7): 743-748.View ArticleGoogle Scholar
  45. Bastien P, Procop GW, Reischl U: Quantitative real-time PCR is not more sensitive than “conventional” PCR. J Clin Microbiol. 2008, 46 (6): 1897-1900.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Oliveira JG, Novais FO, de Oliveira CI, da Cruz Junior AC, Campos LF, da Rocha AV, Boaventura V, Noronha A, Costa JM, Barral A: Polymerase chain reaction (PCR) is highly sensitive for diagnosis of mucosal leishmaniasis. Acta Trop. 2005, 94 (1): 55-59.View ArticlePubMedGoogle Scholar
  47. Palatnik-de-Sousa CB, Paraguai-de-Souza E, Gomes EM, Soares-Machado FC, Luz KG, Borojevic R: Transmission of visceral leishmaniasis by blood transfusion in hamsters. Braz J Med Biol Res. 1996, 29 (10): 1311-1315.PubMedGoogle Scholar
  48. Owens SD, Oakley DA, Marryott K, Hatchett W, Walton R, Nolan TJ, Newton A, Steurer F, Schantz P, Giger U: Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs. J Vet Med Educ. 2001, 219 (8): 1076-1083.Google Scholar
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