ABSTRACT.
Leishmaniasis poses significant public health challenges in endemic regions. Understanding the prevalence of asymptomatic Leishmania infection and identifying risk factors among blood donors is crucial. This study addressed a knowledge gap by evaluating the prevalence of asymptomatic Leishmania infection and pinpointing associated risk factors among blood donors in an endemic area in Thailand and aimed to enhance blood donation safety protocols and reduce the risk of transfusion-transmitted Leishmania infection. A cross-sectional study and a longitudinal follow-up were conducted among 500 blood donors in Trang Province, southern Thailand. A serological test was performed using the direct agglutination test (DAT), and DNA detection was performed using nested polymerase chain reaction (nPCR) to screen for Leishmania infection. Potential risk factors associated with the infection were also assessed. The study identified a 19.0% prevalence of asymptomatic Leishmania infection among blood donors, with nPCR proving more effective in detecting infections (13.0%) than DAT (6.4%). Notably, Leishmania martiniquensis was the predominant species identified, highlighting the local epidemiological profile of Leishmania infection. Furthermore, using multivariate analysis, living in stilt houses was independently associated with Leishmania infection (adjusted odds ratio = 1.85; 95% CI = 1.04–3.28; P = 0.035). A high prevalence of asymptomatic Leishmania infection among blood donors underscores the need for integrating comprehensive Leishmania screening protocols into blood donation processes, particularly in endemic regions. It advocates for using molecular diagnostics to enhance detection accuracy. Furthermore, living in stilt houses as a risk factor emphasizes the importance of environmental management in leishmaniasis control efforts.
INTRODUCTION
Leishmaniasis, a vector-borne disease caused by the protozoan parasites of the genus Leishmania, presents a significant public health challenge with complex transmission dynamics.1,2 Beyond vector transmission, other modes have been increasingly acknowledged, including congenital transmission,2 needle-prick accidents, and the use of contaminated needles among drug users.3 A particularly concerning avenue of transmission is through blood transfusions, which have been demonstrated in animal models4,5 and inferred in human case reports.6,7 The potential for visceral leishmaniasis (VL) transmission via blood transfusion is heightened in endemic regions where asymptomatic carriers among the healthy population are prevalent.7,8 Studies have detected viable Leishmania parasites in stored blood products, suggesting that blood transfusion can be the source of disease propagation to immunocompetent recipients.9,10 As a result, routine screening of blood donors for Leishmania infection is recommended in some endemic areas.10
Recent studies of leishmaniasis in Thailand focused only on high-risk populations, particularly those infected with HIV.11–14 Such studies have disclosed a considerable intersection between immunocompromised conditions and the propensity for leishmaniasis infection. However, one study in northern Thailand reported Leishmania infection within an immunocompetent community and the demographic investigation of this infection.15 Diverging significantly from previous investigations, our inquiry focused on blood donors, a population primarily overlooked in Thailand. The significance of this group is emphasized by the possibility that blood transfusions could serve for disease transmission; failure to assess and manage this risk adequately could result in a considerable public health emergency. This group might also represent the situation of leishmaniasis in the immunocompetent population.
This study investigated the prevalence and associated factors of Leishmania infection among blood donors in Thailand. We aimed to establish the foundation for developing customized screening strategies and enhancing blood safety measures by analyzing the frequency and identifying the factors contributing to blood donors with competent immune systems. The implications of this study extend far beyond its local context. Given that it revealed the epidemiological profile of Leishmania infection among blood donors in one of the endemic areas, southern Thailand, the insights could lead to implementing public health strategies and policy-making processes, thereby enhancing the integrity of the blood supply. This work significantly contributes to the collective global efforts to control Leishmania infection as a public health challenge.
MATERIALS AND METHODS
Study setting.
This study was conducted at the Blood Donation Unit, Trang Hospital, a tertiary medical care center, and a regional hospital with a capacity of 553 beds in Trang Province, southern Thailand, approximately 837 km from Bangkok. The province, known for its coastal geography, experiences a tropical monsoon climate with an average temperature of 27.2°C and annual precipitation of 2,385.1 mm, with September being the peak month for rainfall.14 Trang Hospital is instrumental in providing healthcare services to the local population and caters to individuals from surrounding areas. The hospital’s blood bank is critical in supporting medical services and maintaining a safe blood supply for transfusions. Blood donations at the hospital are screened regularly for infectious diseases such as HIV, hepatitis B and C, and Treponema pallidum, which aligns with the national blood safety standards.16
As a key provider of blood transfusion services in the region, the Blood Donation Unit at Trang Hospital could offer a unique opportunity to investigate the prevalence of asymptomatic Leishmania infection in a population not previously studied in this context. The province’s position and the mobility of its population pose potential risks for introducing and transmitting the parasite, making it a suitable location for this research.
Study design.
This was a cross-sectional study investigating the prevalence of asymptomatic Leishmania infection among blood donors within Trang Province, Thailand.
Sample size calculation.
We applied a single population proportion formula with an anticipated infection rate of 8.7% from a systematic review.7 With a 95% confidence level (z = 1.96) and a 5% margin of error, and incorporating a 5% potential nonresponse rate, the calculation determined that at least 128 participants were required in this study.
Ethics statement.
The present investigation adhered to the ethical guidelines in the Declaration of Helsinki. Ethical approval was obtained from the Institutional Review Board of the Royal Thai Army Medical Department (approval number: S026q/63). Each participant provided written informed consent before they participated in the study. The confidentiality and anonymity of the participants were ensured by assigning unique identification codes to the collected data.
Study population and eligibility criteria.
The study population consisted of individuals who visited Trang Hospital’s Blood Donation Unit from June to August 2022 and met the eligibility criteria for blood donation. These criteria, as outlined by the Thai Red Cross, require donors to be at least 18 years old and in suitable health, as confirmed through routine serological screenings for bloodborne pathogens, including hepatitis B virus (HBV), hepatitis C virus (HCV), HIV, and Treponema pallidum.16 Those who completed the donation process were enrolled in the study.
Data collection.
Informed consent was obtained from all participants; they also completed a self-administered and structured questionnaire to gather sociodemographic information and identify potential risk factors associated with Leishmania infection. The reliability of this questionnaire was previously established through a pretest on a sample of 40 blood donors, achieving a Cronbach’s alpha of 0.85. The questionnaire solicited demographic data, such as age, gender, education, and occupation. Behavioral patterns of interest included intravenous and recreational drug use and travel history, particularly to areas known for Leishmania infection. Health-related questions addressed whether participants had underlying medical conditions such as type 2 diabetes mellitus (T2DM), hypertension, allergies, or a history of blood transfusions.
The study also evaluated environmental exposure factors. Details regarding ownership of pets (i.e., dogs, cats, cattle, and poultry) were collected, along with information about participants’ housing conditions—specifically, living in stilt houses, houses with a corral, houses with a plantation, or houses in the proximity of forests. The frequency of outdoor nighttime activities, such as animal husbandry, agricultural work, or fishing, was also captured to assess the risk of Leishmania transmission. Additionally, preventive practices, including bed nets, were recorded to measure their effectiveness in protecting against vector bites.
Blood sample collection and preparation.
Blood samples were collected in 8-mL EDTA tubes immediately postdonation, using the same needle from the donation procedure to minimize discomfort. The samples underwent centrifugation at 900 × g for 10 minutes to separate plasma and buffy coat for subsequent testing. After centrifugation, the samples were stored at –20°C to preserve their integrity. They remained under these conditions until transported to the Department of Parasitology, Phramongkutklao College of Medicine. This protocol was designed to ensure the highest quality of specimens for accurate detection and characterization.
Detection of Leishmania antibodies.
Antibodies against Leishmania were carried out using the direct agglutination test (DAT; DATkit, KIT Biomedical Research, Amsterdam, the Netherlands). The procedure strictly adhered to the manufacturer’s guidelines. To ensure the accuracy and reliability of the test results, controls were meticulously selected: a positive control was plasma samples from individuals with confirmed cases of leishmaniasis, verified by nested polymerase chain reaction (nPCR). Conversely, a negative control was constituted from plasma samples collected from healthy individuals without history of leishmaniasis. The threshold for a positive result was set at a titer greater than 1:100, in alignment with the manufacturer’s recommendation and a related study employing DAT in the Thai population.11
Detection of Leishmania DNA.
Two hundred microliters of buffy coat samples underwent DNA extraction to detect Leishmania DNA, using the Gen UPTM gDNA Kit (Biotechrabbit, Hennigsdorf, Germany) following the manufacturer’s protocol. The extracted DNA was then eluted into a final volume of 40 µL and subsequently stored at –20°C until further analysis. To amplify Leishmania DNA, the nPCR method was applied, explicitly targeting the internal transcribed spacer 1 (ITS 1) region within the small subunit ribosomal RNA gene of Leishmania. This method, as outlined by Manomat et al., was used to detect Leishmania DNA with high sensitivity and specificity.11 Two rounds of PCR amplification were performed: the first round used outer primers to amplify a larger DNA fragment, followed by a second round with inner primers targeting a specific region within the initial amplicon, thereby increasing the specificity and sensitivity of the assay. This approach allows for detecting low quantities of Leishmania DNA, making it a powerful tool for diagnosing and monitoring infections.
Sequence analysis.
Positive PCR products derived from buffy coat samples were forwarded to U2Bio Co. Ltd. (Seoul, South Korea) for detailed sequence analysis. Upon receipt, each sample’s sequencing output was inspected using BioEdit version 7.0.1 (Ibis Therapeutics, Carlsbad, CA) to validate the chromatograms and confirm the quality of the sequencing results. Subsequently, the sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis to ascertain sequence similarity and ensure the specificity of the detected Leishmania DNA by comparing it against known sequences in the database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), facilitating the accurate identification of the Leishmania species present in each sample.
Definition.
Asymptomatic Leishmania infection: an individual was considered to have an asymptomatic Leishmania infection if they did not show any clinical symptoms commonly associated with cutaneous leishmaniasis (CL) or VL test positive for the infection. Detection was achieved through DAT or nPCR. It is essential to note that seropositivity merely indicates the presence of antibodies against Leishmania and can be found in both symptomatic and asymptomatic individuals, reflecting exposure to the parasites.
Positive Leishmania infection: a positive case of Leishmania infection was defined as a participant who tested positive for the parasite using either DAT or nPCR. These tests are conducted on samples extracted from the blood’s buffy coat component. Identifying Leishmania DNA or antibodies in the buffy coat confirms infection, regardless of symptoms.
STATISTICAL ANALYSES
In this study, the prevalence of Leishmania infection among blood donors was calculated by dividing the number of Leishmania-positive individuals by the total number of study participants. Data collected from participants was securely entered into a computerized database protected by password access to ensure data integrity and confidentiality. Descriptive statistics provided a comprehensive overview of the participant demographics and clinical characteristics. These included calculating frequencies and proportions for categorical variables and means and standard deviations for continuous variables. χ2 or Fisher’s exact tests were applied to explore associations between categorical variables and Leishmania infection based on the data distribution’s adequacy and the dataset’s size.
Identifying risk factors for Leishmania infection was approached through univariate and multivariate logistic regression analyses. Variables statistically significant in previous research or exhibiting a P <0.25 in the univariate analysis were advanced to multivariate logistic regression. This step was crucial for adjusting for potential confounders and determining independent predictors of Leishmania infection. The outcomes of these analyses were expressed as odds ratios (ORs) and 95% CIs at a P <0.05 in the multivariate logistic regression, highlighting factors independently associated with Leishmania infection. The statistical processing and analysis of the collected data were performed using STATA software, version SE18 (Stata Corporation, College Station, TX).
RESULTS
The prevalence of Leishmania infection and its associated factors among blood donors in Trang Province, Thailand, among 500 participants, are presented in Table 1. Of these, 95 individuals tested positive for Leishmania, showing a prevalence rate of 19.0% (95% CI = 15.6–22.7). The questionnaire covered demographic, behavioral, and environmental factors to identify potential correlations with Leishmania infection.
Table 1.
Baseline characteristics and associated factors with Leishmania infection among blood donors, Trang Province, southern Thailand
| Characteristics | Negative (n = 405) | Positive (n = 95) | Total (N = 500) | P-Value |
|---|---|---|---|---|
| Demographic factors | ||||
| Gender | 0.561 | |||
| Male | 201 (49.6%) | 44 (46.3%) | 245 (49.0%) | |
| Female | 204 (50.4%) | 51 (53.7%) | 255 (51.0%) | |
| Age | ||||
| Mean (SD) | 37.5 (10.7) | 35.6 (11.9) | 37.1 (10.9) | 0.387 |
| Max–min | 18–65 | 18–63 | 18–65 | |
| Education level | 0.250 | |||
| Primary school | 25 (6.2%) | 5 (5.3%) | 30 (6.0%) | |
| Secondary school | 142 (35.1%) | 42 (44.2%) | 184 (36.8%) | |
| Bachelor’s degree and higher | 238 (58.7%) | 48 (50.5%) | 286 (57.2%) | |
| Occupation | 0.137 | |||
| Merchant | 61 (15.1%) | 8 (8.4%) | 69 (13.8%) | |
| Farmer | 80 (19.8%) | 15 (15.8%) | 95 (19.0%) | |
| Government/officer | 168 (41.5%) | 39 (41.0%) | 207 (41.4%) | |
| Employee | 48 (11.8%) | 15 (15.8%) | 63 (12.6%) | |
| Student | 48 (11.8%) | 18 (18.9%) | 66 (13.2%) | |
| Behavioral factors | ||||
| Intravenous drug user | 0.526 | |||
| No | 403 (99.5%) | 94 (98.9%) | 497 (99.4%) | |
| Yes | 2 (0.5%) | 1 (1.1%) | 3 (0.6%) | |
| Recreational drug user | 0.835 | |||
| No | 395 (97.5%) | 93 (97.9%) | 488 (97.6%) | |
| Yes | 10 (2.5%) | 2 (2.1%) | 12 (2.4%) | |
| History of travel aboard | 0.467 | |||
| No | 383 (94.6%) | 88 (92.6%) | 471 (94.2%) | |
| Yes | 22 (5.4%) | 7 (7.4%) | 29 (5.8%) | |
| Health status | ||||
| Presence of underlying conditions | 0.406 | |||
| No | 373 (92.1%) | 85 (89.5%) | 458 (91.6%) | |
| Yes | 32 (7.9%) | 10 (10.5%) | 42 (8.4%) | |
| Type 2 diabetes mellitus | 0.493 | |||
| No | 403 (99.5%) | 95 (100.0%) | 498 (99.6%) | |
| Yes | 2 (0.5%) | 0 | 2 (0.4%) | |
| Hypertension | 0.479 | |||
| No | 397 (98.0%) | 92 (96.8%) | 489 (97.8%) | |
| Yes | 8 (2.0%) | 3 (3.2%) | 11 (2.2%) | |
| Allergy | 0.407 | |||
| No | 391 (96.5%) | 90 (94.7%) | 481 (96.2%) | |
| Yes | 14 (3.5%) | 5 (5.3%) | 19 (3.8%) | |
| History of received blood transfusion | 0.569 | |||
| No | 366 (90.4%) | 84 (88.4%) | 450 (90.0%) | |
| Yes | 39 (9.6%) | 11 (11.6%) | 50 (10.0%) | |
| Environmental factors | ||||
| Animal exposure | ||||
| Pet ownership | 0.151 | |||
| No | 151 (37.3%) | 43 (45.3%) | 194 (38.8%) | |
| Yes | 254 (62.7%) | 52 (54.7%) | 306 (61.2%) | |
| Dog ownership | 0.038* | |||
| No | 230 (56.8%) | 65 (68.4%) | 295 (59.0%) | |
| Yes | 175 (43.2%) | 30 (31.6%) | 205 (41.0%) | |
| Cat ownership | 0.572 | |||
| No | 264 (65.2%) | 59 (62.1%) | 323 (64.6%) | |
| Yes | 141 (34.8%) | 36 (37.9%) | 177 (35.4%) | |
| Cattle ownership | 0.114 | |||
| No | 380 (93.8%) | 93 (97.9%) | 473 (94.6%) | |
| Yes | 25 (6.2%) | 2 (2.1%) | 27 (5.4%) | |
| Poultry ownership | 0.941 | |||
| No | 357 (88.2%) | 84 (88.4%) | 441 (88.2%) | |
| Yes | 48 (11.8%) | 11 (11.6%) | 59 (11.8%) | |
| Housing conditions | ||||
| Living in stilt houses | 0.070 | |||
| No | 339 (83.7%) | 72 (75.8%) | 411 (82.2%) | |
| Yes | 66 (16.3%) | 23 (24.2%) | 89 (17.8%) | |
| A house with a corral | 0.811 | |||
| No | 382 (94.3%) | 89 (93.7%) | 471 (94.2%) | |
| Yes | 23 (5.7%) | 6 (6.3%) | 29 (5.8%) | |
| A house with a plantation | 0.719 | |||
| No | 351 (86.7%) | 81 (85.3%) | 432 (86.4%) | |
| Yes | 54 (13.3%) | 14 (14.7%) | 68 (13.6%) | |
| A house adjacent to a forest | 0.196 | |||
| No | 364 (89.9% | 81 (85.3%) | 445 (89.0%) | |
| Yes | 41 (10.1%) | 14 (14.7%) | 55 (11.0%) | |
| Outdoor activity | ||||
| Nighttime animal raising | 0.695 | |||
| No | 384 (94.8%) | 91 (95.8%) | 475 (95.0%) | |
| Yes | 21 (5.2%) | 4 (4.2%) | 25 (5.0%) | |
| Nighttime farming | 0.604 | |||
| No | 321 (79.3%) | 73 (76.8%) | 394 (78.8%) | |
| Yes | 84 (20.7%) | 22 (23.2%) | 106 (21.2%) | |
| Nighttime fishing | 0.944 | |||
| No | 396 (97.8%) | 93 (97.9%) | 489 (97.8%) | |
| Yes | 9 (2.2%) | 2 (2.1%) | 11 (2.2%) | |
| Preventive measure | ||||
| Bed net use | 0.911 | |||
| No | 220 (54.3%) | 51 (53.7%) | 271 (54.2%) | |
| Yes | 185 (45.7%) | 44 (46.3%) | 229 (45.8%) | |
Using the χ2 test, significantly different at P <0.05.
The demographic profile of the study population revealed a balanced gender distribution, with males 49.0% and females 51.0%. The infection rate did not significantly differ between genders (P = 0.561). Age analysis indicated a broad spectrum, with an overall mean age of 37.1 years. The difference in mean ages between negative and positive Leishmania infection cases was not statistically significant (P = 0.387). Educational attainment and occupational status varied among the participants. However, neither was significantly associated with Leishmania infection, with education levels (P = 0.250) and occupation types (P = 0.137) showing no discernible pattern in infection distribution.
Regarding behavioral factors, intravenous drug use and recreational drug use were relatively rare behaviors among participants, contributing insignificantly to a risk of Leishmania infection, with P = 0.526 and 0.835, respectively. In addition, 29 (5.8%) of participants identified a history of travel abroad, which did not correlate with the infection (P = 0.467).
Underlying health conditions such as T2DM and hypertension were present in a minority of the population. They did not significantly affect the likelihood of Leishmania infection, with P = 0.493 and 0.479, respectively. This observation implied that these conditions alone did not elevate the risk of Leishmania transmission.
For environmental factors, dog ownership, in particular, was significantly linked to a higher infection risk, with 205 (41.0%) participants owning dogs, showing a statistically significant association (P = 0.038). Participation in nighttime outdoor activities did not significantly correlate with infection rates.
Adopting preventive measures, such as using bed nets, was widespread among participants. Nevertheless, it did not significantly alter the risk of Leishmania infection (P = 0.911), suggesting that additional measures may be necessary for effective prevention.
Leishmania infection and species identification in blood donors.
Table 2 shows the distribution and detection of Leishmania infection using DAT and nPCR. Of 500 samples, 95 (19.0%, 95% CI = 15.6–22.7) were positive for Leishmania infection; nPCR identified 65 cases (13.0%, 95% CI = 10.1–16.3), with the majority being Leishmania martiniquensis (64 cases, 98.5%). Only one case was attributed to the Leishmania donovani complex. The DAT revealed 32 positive cases (6.4%, 95% CI = 4.4–8.8), with the most common titers being 1:200 and 1:100. Notably, two individuals were positive using both methods, confirming cases of L. martiniquensis infection with DAT titers of 1:100 and 1:200.
Table 2.
Detection methods of Leishmania infection among 95 positive cases using DAT or nPCR at baseline recruitment
| Method | No. of Positive Cases | Species Identification (n) | DAT Titer (n) |
|---|---|---|---|
| DAT only | 30 | 1:100 (12) | |
| 1:200 (13) | |||
| 1:400 (4) | |||
| 1:800 (1) | |||
| nPCR only | 63 | L. martiniquensis (62) | |
| L. donovani complex (1) | |||
| DAT and nPCR | 2 | L. martiniquensis (2) | 1:100 (1) |
| 1:200 (1) | |||
| Total | 95 |
DAT = direct agglutination test; nPCR = nested polymerase chain reaction.
Participant recruitment and longitudinal monitoring of Leishmania infection.
Figure 1 illustrates the study’s participant flow and follow-up of Leishmania infection cases during the baseline enrollment from June to August 2022. The first follow-up, conducted from November to December 2022 among the initially positive cases, included 66 participants due to loss to follow-up. In this cohort, 57 tested negative, and nine remained positive, with eight cases positive by DAT only and one by nPCR only. The second follow-up took place in July and August 2023 and involved six of the previously positive cases and a further loss to follow-up. All six cases tested positive again, each positive by DAT only. The flow chart demonstrates the retention and conversion rates across the study period, providing insights into the persistence of Leishmania infection over time.
Figure 1.
Population flow chart showing participant recruitment and follow-up of Leishmania infection cases. DAT = direct agglutination test; nPCR = nested polymerase chain reaction.
Longitudinal analysis of persistently positive Leishmania cases in blood donors.
Table 3 presents the longitudinal laboratory profiles for nine cases that consistently tested positive for Leishmania infection over three time points: baseline (June–August 2022), first follow-up (November–December 2022), and second follow-up (July–August 2023). The detection methods used were DAT-and nPCR.
Table 3.
Longitudinal laboratory profiles for Leishmania Infection in nine persistently positive cases using DAT or nPCR
| Case No. | Baseline | Follow-Up 1 | Follow-Up 2 | |||
|---|---|---|---|---|---|---|
| (June–Aug 2022) | (Nov–Dec 2022) | (July–Aug 2023) | ||||
| DAT | nPCR | DAT | nPCR | DAT | nPCR | |
| 1 | 1:100 | Negative | 1:200 | Negative | 1:3,200 | Negative |
| 2 | 1:100 | Negative | 1:100 | Negative | Lost to follow-up | |
| 3 | 1:200 | Negative | Neg | L. donovani complex | Lost to follow-up | |
| 4 | 1:200 | Negative | 1:200 | Negative | Lost to follow-up | |
| 5 | 1:200 | Negative | 1:200 | Negative | 1:3,200 | Negative |
| 6 | 1:200 | Negative | 1:3,200 | Negative | 1:3,200 | Negative |
| 7 | 1:200 | Negative | 1:3,200 | Negative | 1:200 | Negative |
| 8 | 1:400 | Negative | 1:6,400 | Negative | 1:3,200 | Negative |
| 9 | Negative | L. martiniquensis | 1:6,400 | Negative | 1:3,200 | Negative |
DAT = direct agglutination test; nPCR = nested polymerase chain reaction.
At baseline, the DAT titers ranged from 1:100 to 1:400, with one individual testing negative (Neg) using DAT but positive for L. martiniquensis using nPCR. One case was identified as L. donovani complex using nPCR during the first follow-up. In contrast, the others were negative using nPCR and exhibited DAT titers from 1:200 to 1:6,400. By the second follow-up, some individuals were lost to follow-up, but among those who remained, DAT titers ranged from 1:200 to 1:3,200, all with negative nPCR results. Table 3 illustrates the persistence of Leishmania antibodies over time as detected by DAT, despite the absence of DNA detection by nPCR in later follow-ups, suggesting potential seropositivity without active infection. None of the infected subjects developed the disease during the 1-year study period.
Associated risk factors for Leishmania infection among blood donors.
Table 4 demonstrates the factors associated with Leishmania infection among blood donors in Trang Province, using univariate and multivariate logistic regression. Females had a slightly higher rate of infection (20%) compared with males (18%); gender was not a significant predictor of Leishmania infection in the multivariate analysis with adjusted OR (aOR) = 0.98; 95% CI = 0.60–1.58; P = 0.938). Age also did not significantly predict infection after adjustment (aOR = 0.98; 95% CI = 0.96–1.01; P = 0.325). Different occupations were also compared. Being a student was significantly associated with infection in the univariate analysis (crude OR = 2.85; P = 0.024), but this association was not found in the multivariate analysis (aOR = 2.45; 95% CI = 0.89–6.76; P = 0.082). Behavioral factors, such as intravenous and recreational drug use, were not significantly associated with Leishmania infection. Similarly, a history of travel abroad was not a significant predictor. The analysis also considered health status factors, such as underlying conditions and a history of received blood transfusions. Neither of these factors was significantly associated with infection in the multivariate analysis. Among environmental factors, dog ownership and specific housing conditions were assessed. Only one factor, living in stilt houses, significantly predicted the infection using the multivariate model (aOR = 1.85; 95% CI = 1.04–3.28; P = 0.035). Other housing conditions and outdoor activities were not significantly associated with the risk of infection. Preventive measures, specifically bed net use, did not significantly alter the risk of infection (aOR = 1.15; 95% CI = 0.71–1.87; P = 0.573).
Table 4.
Univariate and multivariate logistic regression analysis of factors associated with Leishmania infection among blood donors in Trang Province
| Characteristics | Negative | Positive | Total | cOR | 95% CI | P-Value | aOR | 95% CI | P-Value |
|---|---|---|---|---|---|---|---|---|---|
| n = 405 (%) | n = 95 (%) | N = 500 (%) | |||||||
| Demographic factors | |||||||||
| Sex | |||||||||
| Male | 201 (82.9) | 44 (18.0) | 245 (49.0) | 1.00 | 1.00 | ||||
| Female | 204 (80.0) | 51 (20.0) | 255 (51.0) | 1.14 | 0.73–1.79 | 0.561 | 0.98 | 0.60–1.58 | 0.938 |
| Age, mean (SD) | 37.5 (10.7) | 35.6 (11.9) | 37.1 (10.9) | 0.98 | 0.96–1.00 | 0.141 | 0.98 | 0.96–1.01 | 0.325 |
| Occupation | |||||||||
| Merchant | 61 (88.4) | 8 (11.6) | 69 (13.8) | 1.00 | 1.00 | ||||
| Farmer | 80 (84.2) | 15 (15.8) | 95 (19.0) | 1.43 | 0.57–3.59 | 0.447 | 1.31 | 0.48–3.62 | 0.596 |
| Government/officer | 168 (81.2) | 39 (18.8) | 207 (41.4) | 1.77 | 0.78–3.99 | 0.170 | 1.67 | 0.72–3.84 | 0.228 |
| Employee | 48 (76.2) | 15 (23.8) | 63 (12.6) | 2.38 | 0.93–6.08 | 0.070 | 2.47 | 0.94–6.47 | 0.066 |
| Student | 48 (72.7) | 18 (27.3) | 66 (13.2) | 2.85 | 1.14–7.14 | 0.024* | 2.45 | 0.89–6.76 | 0.082 |
| Behavioral factors | |||||||||
| Intravenous drug user | |||||||||
| No | 403 (81.1) | 94 (18.9) | 497 (99.4) | 1.00 | 1.00 | ||||
| Yes | 2 (66.7) | 1 (33.3) | 3 (0.6) | 2.14 | 0.19–23.89 | 0.535 | 2.97 | 0.11–78.15 | 0.514 |
| Recreational drug user | |||||||||
| No | 395 (80.9) | 93 (19.1) | 488 (97.6) | 1.00 | 1.00 | ||||
| Yes | 10 (83.3) | 2 (16.7) | 12 (2.4) | 0.85 | 0.18–3.94 | 0.835 | 0.58 | 0.07–5.00 | 0.621 |
| History of travel aboard | |||||||||
| No | 383 (81.3) | 88 (18.7) | 471 (94.2) | 1.00 | 1.00 | ||||
| Yes | 22 (75.9) | 7 (24.1) | 29 (5.8) | 1.38 | 0.57–3.34 | 0.469 | 1.53 | 0.61–3.89 | 0.367 |
| Health status | |||||||||
| Presence of underlying conditions | |||||||||
| No | 373 (81.4) | 85 (18.6) | 458 (91.6) | 1.00 | 1.00 | ||||
| Yes | 32 (76.2) | 10 (23.8) | 42 (8.4) | 1.37 | 0.65–2.89 | 0.408 | 1.52 | 0.68–3.40 | 0.309 |
| History of blood transfusion | |||||||||
| No | 366 (81.3) | 84 (18.7) | 450 (90.0) | 1.00 | 1.00 | ||||
| Yes | 39 (78.0) | 11 (22.0) | 50 (10.0) | 1.23 | 0.60–2.49 | 0.569 | 1.31 | 0.62–2.76 | 0.472 |
| Environmental factors | |||||||||
| Animal exposure | |||||||||
| Dog ownership | |||||||||
| No | 230 (78.0) | 65 (22.0) | 295 (59.0) | 1.00 | 1.00 | ||||
| Yes | 175 (85.4) | 30 (14.6) | 205 (41.0) | 0.59 | 0.38–0.97 | 0.039* | 0.65 | 0.29–1.06 | 0.082 |
| Housing conditions | |||||||||
| Living in stilt houses | |||||||||
| No | 339 (82.5) | 72 (17.5) | 411 (82.2) | 1.00 | 1.00 | ||||
| Yes | 66 (74.2) | 23 (25.8) | 89 (17.8) | 1.64 | 0.96–2.81 | 0.071 | 1.85 | 1.04–3.28 | 0.035† |
| A house with a corral | |||||||||
| No | 382 (81.1) | 89 (18.9) | 471 (94.2) | 1.00 | 1.00 | ||||
| Yes | 23 (79.3) | 6 (20.7) | 29 (5.8) | 1.12 | 0.44–2.83 | 0.811 | 1.07 | 0.38–3.00 | 0.891 |
| A house with a plantation | |||||||||
| No | 351 (81.2) | 81 (18.7) | 432 (86.4) | 1.00 | 1.00 | ||||
| Yes | 54 (79.4) | 14 (20.6) | 68 (13.6) | 1.12 | 0.59–2.12 | 0.720 | 1.23 | 0.61–2.44 | 0.562 |
| A house with an adjacent forest | |||||||||
| No | 364 (81.8) | 81 (18.2) | 445 (89.0) | 1.00 | 1.00 | ||||
| Yes | 41 (74.5) | 14 (25.4) | 55 (11.0) | 1.53 | 0.79–2.95 | 0.199 | 1.55 | 0.78–3.08 | 0.211 |
| Outdoor activity | |||||||||
| Nighttime animal raising | |||||||||
| No | 384 (80.8) | 91 (19.2) | 475 (95.0) | 1.00 | 1.00 | ||||
| Yes | 21 (84.0) | 4 (16.0) | 25 (5.0) | 0.8 | 0.27–2.39 | 0.695 | 0.82 | 0.23–2.87 | 0.757 |
| Nighttime farming | |||||||||
| No | 321 (81.5) | 73 (18.5) | 394 (78.8) | 1.00 | 1.00 | ||||
| Yes | 84 (79.2) | 22 (20.8) | 106 (21.2) | 1.15 | 0.67–1.96 | 0.604 | 1.49 | 0.75–2.98 | 0.253 |
| Nighttime fishing | |||||||||
| No | 396 (81.0) | 93 (19.0) | 489 (97.8) | 1.00 | 1.00 | ||||
| Yes | 9 (81.8) | 2 (18.2) | 11 (2.2) | 0.94 | 0.20–4.45 | 0.944 | 1.02 | 0.18–5.71 | 0.984 |
| Preventive measure | |||||||||
| Bed net use | |||||||||
| No | 220 (81.2) | 51 (18.8) | 271 (54.2) | 1.00 | 1.00 | ||||
| Yes | 185 (80.8) | 44 (19.2) | 229 (45.8) | 1.02 | 0.65–1.60 | 0.911 | 1.15 | 0.71–1.87 | 0.573 |
aOR = adjusted odds ratio; cOR = crude odd ratio.
Using univariate analysis, significantly different at P <0.05.
Using multivariate analysis, significantly different at P <0.05 after adjusting for demographic factors (sex, age, occupation), animal exposure (dog ownership), housing conditions (living in stilt houses, a house with a corral, a house with a plantation, a house with an adjacent forest), outdoor activity (nighttime animal raising, nighttime agriculture work), and preventive measure (bed net use).
DISCUSSION
The revelation of a 19% prevalence of asymptomatic Leishmania infection among blood donors in Trang Province, southern Thailand, marks a pioneering investigation into an area that has previously seen limited research. This finding highlights the potential for transmission of Leishmania within healthy populations. The variability in the prevalence of asymptomatic Leishmania infection among blood donors reflects the diverse epidemiological landscape of the disease across different geographies. It becomes even more critical when considering the variability in diagnostic methods observed in global studies.7,17 European data demonstrated substantial variation, from a seroprevalence of 4.1% in Italy as detected by the indirect hemagglutination test in 1991,7 to a higher 13.4% in France in 1999 using a Western blot (WB) technique targeting the L. infantum antigen fraction.18 Spain reported a 3.1% prevalence among a significant cohort of 1,437 blood donors in 2008.19 The DAT was notably effective in diagnosing VL in East Africa; it was less efficient than the rK39 rapid diagnostic ELISA for detecting asymptomatic infections, likely due to diminished antibody production in such cases.7 This pattern of variability extended beyond Europe, with Ethiopia reporting a prevalence of 15% in Metema, a known VL-endemic area, and 4.2% in Gondar, a nonendemic region.6 Brazil illustrated this global disparity, where prevalence rates peaked at 15.6% in 2013, later reducing to 5.4% among 700 donors tested in Salvador in 2014.7,20 The recent findings from Portugal, indicating a prevalence of 4.8% in 2023,21 Asian countries also exhibited a broad spectrum of prevalence rates, underscoring the widespread epidemiological diversity of Leishmania infection. For instance, Bangladesh reported a notably low prevalence of 0.25% in 2013 using the rK39 strip test.22 In contrast, Nepal’s application of DAT in 2016 identified a 1% prevalence.23 Meanwhile, Iran in 2022 reported that 24 (2.8%) individuals were seropositive for VL using DAT, and a striking 388 (45%) were positive by kinetoplast DNA-PCR,17 emphasizing the variable burden of Leishmania infection across different Asian settings.
The differences in reported prevalence rates can be attributed to several factors, including regional endemicity levels, species of Leishmania, genetic variation, geographic distribution,24 population demographics, age distribution, and the diagnostic methods used to detect asymptomatic infections.7 The poor correlation between different diagnostic tests further emphasizes the need for a multifaceted testing approach to enhance sensitivity and specificity. In this study, the high prevalence rate of 19% presents the epidemiological complexity associated with Leishmania infection, reinforcing the importance of screening methods in blood donation settings, particularly in regions with varying endemicity levels.
The choice of diagnostic method for Leishmania screening in blood donors is crucial.17 Our study’s integration of DAT and nPCR was designed to address the typically weak correlation between the two tests.10,20,25,26 DAT, a serological assay, detected antibodies against Leishmania, indicating a 6.4% prevalence. However, the reliance on the immune response can lead to underestimations of prevalence, especially in asymptomatic cases with insufficient antibody levels for detection.6 In contrast, nPCR, focusing on parasite DNA, marked a 13% prevalence, suggesting greater sensitivity in identifying asymptomatic carriers. This method’s ability to detect low parasite DNA levels makes it invaluable for minimizing transfusion-transmitted Leishmania risks,6,25
This study observed variations in DAT titers across the cases, particularly significant increases in some individuals. This could be attributed to the immune response to persistent or repeated exposure to Leishmania antigens. The nPCR results being consistently negative in the follow-up period suggests that antibodies were produced, indicating exposure or past infection; active parasitemia was undetected. This highlights the complexity of interpreting serological tests in the context of Leishmania infections, where antibody levels may remain elevated long after the resolution of active disease.
The concurrent identification of two cases through DAT and nPCR underscores the intricate nature of diagnosing Leishmania infections and the necessity of employing a multimodal diagnostic framework to improve sensitivity. Nonetheless, adopting nPCR for routine screening in blood banks necessitates carefully evaluating factors like technological accessibility, cost, and scalability. Moreover, integrating nPCR into blood donation processes would require comprehensive protocols and guidelines for managing detected asymptomatic infections.25
Our research into the species-specific detection of Leishmania among blood donors in Trang Province, Thailand, offers substantial epidemiological insight, notably through the predominant species of L. martiniquensis, identified in 64 of 65 positive cases using nPCR. The results contrast with the global landscape, where species such as L. infantum and L. donovani are frequently implicated in human cases, pointing toward unique ecological and transmission dynamics of Leishmania infection within Southeast Asian countries.9,20,21,27–31 L. martiniquensis’s prevalence in our cohort suggests different vector–host interactions characteristic of the region, distinct from the zoonotic and anthroponotic cycles driven by L. infantum and L. donovani in other endemic areas.21,27,28 This finding emphasizes the ecological specificity of L. martiniquensis transmission and the importance of localized surveillance to study the epidemiological patterns of this disease. Interestingly, our research did not detect Leishmania orientalis in the blood donor population, a species previously identified in HIV/AIDS patients in the region.11,13,14 This absence might suggest that the transmission dynamics of L. orientalis differ, possibly due to weaker evasion of host immunity compared with L. martiniquensis. This distinction underscores the complex interplay among different Leishmania species, host immune responses, and epidemiological patterns, necessitating targeted studies to unravel these relationships. Furthermore, identifying an L. donovani complex case within our study signals the potential for imported cases or previous transmission dynamics in Thailand. The role of human movement in the dispersion of Leishmania species underscores the necessity for careful epidemiological monitoring. Future research should focus on unraveling the ecological and social factors that influence the distribution of Leishmania species, enhancing our ability to control Leishmania infection in endemic and nonendemic settings.
In this study, a significant environmental factor, such as living in a traditional Thai stilt house, was associated with L. martiniquensis infection. The stilts raise the house above the soil’s surface to protect it from seasonal flooding and provide air ventilation underneath the floor. The area underneath the floor was used for household activities and relaxation and could also be favorable for vector habitats and bites. This finding is consistent with our previous study in Trang Province in 2017, reporting that living in stilt houses was a predictor of Leishmania infection in the southern area of Thailand.14 Due to the zoonotic transmission nature of L. martiniquensis, we specifically focused on animal ownership, especially for dogs and cattle. Univariate analysis showed that dog ownership was significantly associated with infection (P <0.039). When adjusting the confounding factors using multivariate analysis, dogs were not an independently associated risk factor for the infection (P <0.082); this could be due to the influence of other environmental or behavioral characteristics that might have a more direct role in transmission. Although suitable for vector habitats, the under-floor area of stilt houses might expose all residents to vectors, regardless of dog ownership. Thus, the specific behavior of using the space under the house or other socioenvironmental conditions related to stilt house living could play a crucial role in exposure to L. martiniquensis, diluting the apparent effect of dog ownership in the multivariate analysis.
Certain environmental conditions, such as proximity to animal shelters or organic waste, increase the risk of vector presence, and thus the transmission of Leishmania.32 Contact with livestock and residing in rural areas were associated with Leishmania infection in the other regions,8,21,28 This variation could be due to differing vector behaviors or human–animal interaction unique to southeast Asia.24 The lack of significance for these factors in our research may point to other unexplored elements influencing transmission in Thailand.
Managing blood donors found positive for Leishmania infection necessitates a nuanced approach, especially the results of our longitudinal findings. In our study, of the nine initially positive cases at baseline, eight were positive by DAT alone and one by nPCR only. During the first follow-up, seven DAT-positive cases remained positive, with one case transitioning from DAT-positive to nPCR-positive, illustrating the dynamic nature of Leishmania infection markers. The case initially detected by nPCR alone was no longer nPCR positive but was DAT positive. One year later, six cases remained in the study by the second follow-up, all testing positive solely via DAT. This consistent seropositivity, absent concurrent nPCR confirmation, indicated a sustained immune response without direct evidence of active infection. It raises critical questions regarding the optimal management of such donors and the blood they donate.
Regarding blood bank protocols, it may be prudent to defer donors who remain seropositive over time because they potentially harbor the parasite, posing a risk for transmission through transfusion, especially to immunocompromised recipients. Continuous DAT positivity warrants a cautious approach; these donors might be temporarily excluded from donation until further confirmatory tests negate active infection risks.25 Given that nPCR detected the DNA of Leishmania and was thus indicative of an active infection, its negative result in the follow-up of donors initially positive by DAT suggested that these donors might not have an active infection at the time of follow-up. However, because they demonstrated an immune response, as shown by DAT, they could have either cleared the infection or harbored the parasite in a controlled state that could not yield detectable DNA.9,25 Considering the clinical management, asymptomatic donors with persistent DAT positivity but negative nPCR results might not require antileishmanial treatment but should be monitored over time. Regular monitoring could involve repeated serological and molecular tests to ensure that any potential progression to active disease is promptly detected and managed.6,7,25 For immunocompromised populations, such as patients with HIV, the management of Leishmania-positive blood donors takes on additional protocol. Given the higher risk of VL after transfusion of contaminated blood in these patients, stringent screening protocols must be implemented.9,10 A study in midwestern Brazil revealed the viability of Leishmania parasites in blood donors using PCR amplification of parasitic rRNA segments.33 In addition, assessing the viability and transmissibility of Leishmania in stored blood products from seropositive donors was demonstrated.6,9 This approach is supported by evidence from a Brazilian study in which infected blood recipients exhibited seroconversion but did not progress to active VL within 6 months.29 Nonetheless, the latent risk persists, especially over extended periods or without consistent follow-up.10 Preemptive measures, including more sensitive screening protocols for Leishmania infections in blood donors, are warranted for high-risk groups.
These findings underscore the need to evaluate thoroughly the potential infectivity of blood from donors for future research and refinement of blood donation protocols.6 Furthermore, exploring pathogen-reduction techniques in the blood supply could add safety,29 particularly in areas endemic to leishmaniasis. Ultimately, the goal is safeguarding the blood supply without unnecessarily reducing the donor pool.
CONCLUSION
In conclusion, this pioneering study in Trang Province, Thailand, revealed the significant prevalence of asymptomatic Leishmania infection among blood donors, uncovering a 19% infection rate that underscores the need for enhanced screening protocols in blood banks, especially in endemic regions. By employing both DAT and nPCR, the research reported a predominant L. martiniquensis infection. Moreover, housing conditions, explicitly living in stilt houses, as a risk factor for infection, highlight the intricate interplay between environmental factors and the transmission of Leishmania. It emphasizes implementing informed blood safety protocols to protect donors and recipients, particularly those who are immunocompromised, from potential transfusion-transmitted infections.
ACKNOWLEDGMENTS
We are grateful to those who participated in the successful completion of this study. Special thanks to all the participants whose willingness to contribute has been invaluable to our research. Their participation has facilitated an understanding of Leishmania infection among blood donors and paved the way for future interventions to improve blood safety and public health outcomes.
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