Diagnosis Of Human Visceral Leishmaniasis Research Articles

Comparison of laboratory methods for diagnosis of human visceral leishmaniasis in the Center-West Region of São Paulo State, Brazil

Visceral leishmaniasis (VL) is a neglected disease and represents an important public health problem. The present study aimed to compare the conventional laboratory methods with the molecular method for VL diagnosis in human blood samples. Direct parasitological test of bone marrow aspirate (direct parasitological test), rapid immunochromatographic test (RIT), indirect immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR) were used to evaluate 26 human samples with clinical suspicion of VL. The positivity rates obtained for direct parasitological test, RIT, IFAT and PCR were 80.8%, 80.8%, 73.1% and 84.6%, respectively. PCR had greater positivity and agreement with the other methodologies. Therefore, the use of PCR in the diagnostic routine should be encouraged to complement the serological results.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Real Time PCR-based diagnosis of human visceral leishmaniasis using urine samples.

Diagnosis of visceral leishmaniasis (VL) through the detection of its causative agents namely Leishmania donovani and L. infantum is traditionally based on immunochromatographic tests, microscopy of bone marrow, spleen aspirates, liver or lymph node and differential diagnosis. While the first process has low specificity, the later one carries the risk of fatal hemorrhage. Over the last decade, multiple Polymerase Chain Reaction (PCR) based diagnosis has been developed using blood and urine sample with a varying degree of sensitivity and specificity, an issue worth improving for precision diagnosis. Earlier, we reported a PCR-based diagnosis of L. donovani in peripheral blood using a novel set of PCR primers with absolute specificity. Using the same set of primers and PCR conditions, here we describe diagnosis of L. donovani from urine, for a non-invasive, rapid and safe diagnosis. Diagnosis of VL was carried out using urine samples collected from clinically diagnosed VL patients (n = 23) of Bangladesh in Real Time PCR. Test results were validated by comparing blood samples from the same set of patients. Sensitivity and specificity of this diagnosis was analyzed using retrospective bone marrow samples, collected earlier from confirmed VL patients (n = 19). The method showed 100% sensitivity in detecting L. donovani in urine and corresponding blood and retrospective bone marrow samples, as well as 100% specificity in control groups. A Real Time PCR-based molecular detection system using urine sample is hereafter presented what could be a, non-invasive approach for VL detection with precision and perfection.

Revealing a Novel Antigen Repressor of Differentiation Kinase 2 for Diagnosis of Human Visceral Leishmaniasis in India.

Visceral leishmaniasis (VL) is one of the major global health concerns due to its association with morbidity and mortality. All available diagnostic tools have been, until now, unable to provide a very specific and cost-effective mode of detection for VL globally. Therefore, the design of robust, specific, and commercially translatable diagnostic tests is urgently required. Currently, we are attempting to identify and explore the diagnostic potential of a novel parasite antigen. Repressor of differentiation kinase 2 (RDK2), a serine/threonine kinase, has a versatile role in parasite life cycle progression. However, its role as a diagnostic candidate for VL has not been investigated. Herein, we cloned and over-expressed LdRDK2 and studied the recombinant RDK2 for the diagnosis of human VL using serum and urine samples. In silico analysis predicted that RDK2 is conserved among Leishmania species with the least conservation in humans. RDK2 developed immune-reactive bands with antibodies present in VL patients’ sera, and it demonstrated no cross-reactivity with sera from healthy controls and other diseases. Additionally, RDK2 antigen demonstrated a significant reactivity with IgG antibodies of VL patients’ sera, with 78% sensitivity and 86.67% specificity as compared to healthy controls and other diseases. Furthermore, we evaluated its utility for non-invasive diagnosis of VL using patients’ urine samples and found 93.8% sensitivity and 85.7% specificity. RDK2 was found to have better sensitivity and treatment response in patients’ urine compared to serum samples, indicating its role as a promising point of care (POC) antigen. In a nutshell, we explored the role of RDK2 as a potential diagnostic marker for VL in both invasive and non-invasive modes as well as its utility as a promising POC antigen for treatment response cases.

Development and Clinical Evaluation of Serum and Urine-Based Lateral Flow Tests for Diagnosis of Human Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a fatal parasitic infection, is categorized as being neglected among tropical diseases. The use of conventional tissue aspiration for diagnosis is not possible in every setting. The immunochromatography-based lateral flow assay (LFA) has attracted attention for a long time due to its ability to give results within a few minutes, mainly in resource-poor settings. In the present study, we optimized and developed the LFA to detect anti-Leishmania antibodies for VL diagnosis. The performance of the developed test was evaluated with serum and urine samples of Indian VL patients and Brazilian sera. The new test exploits well-studied and highly-sensitive purified antigens, LAg isolated from Leishmania donovani promastigotes and protein G conjugated colloidal-gold as a signal reporter. The intensity of the bands depicting the antigen–antibody complex was optimized under different experimental conditions and quantitatively analyzed by the ImageJ software. For the diagnosis of human VL in India, LFA was found to be 96.49% sensitive and 95% specific with serum, and 95.12% sensitive and 96.36% specific with urine samples, respectively. The sensitivity and specificity of LFA were 88.57% and 94.73%, respectively, for the diagnosis of Brazilian VL using patients’ sera infected with Leishmania infantum. LFA is rapid and simple to apply, suitable for field usage where results can be interpreted visually and particularly sensitive and specific in the diagnosis of human VL. Serum and urine LFA may improve diagnostic outcomes and could be an alternative for VL diagnosis in settings where tissue aspiration is difficult to perform.

Development of a Multi-Epitope Recombinant Protein for the Diagnosis of Human Visceral Leishmaniasis.

Background:Iran is one of the endemic areas of Mediterranean Visceral Leishmaniasis, a disease caused by Leishmania infantum. In this work, we examined whether Proteína quimérica 10 (PQ10) recombinant protein is suitable for immunological diagnosis of human visceral leishmaniasis.Methods:The study was carried out in Tarbiat Modares University during 2016–2018. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. Sequencing with proper primers was done, the expression, optimization of expression and protein purification were performed, and the purified recombinant protein was confirmed by western blot. The efficacy of PQ10 for serodiagnosis was evaluated with 50 positive and 50 negative serum samples, which confirmed by the direct agglutination test and collected from individuals living in the visceral leishmaniasis endemic areas of Iran. ELISA was performed with the PQ10 recombinant protein.Results:The 95% CI sensitivity of ELISA that was evaluated with sera from naturally infected individuals was 84%. The 95% CI specificity value of the ELISA determined with sera from healthy individuals (50 serum samples) and from individuals with other infectious diseases was 82%. The 95% CI positive predictive value (PPV) and negative predictive value (NPV) were exterminated 82.35% and 83.67%, respectively.Conclusion:We have used a recombinant synthetic protein to improve serodiagnosis of human visceral leishmaniasis. PQ10 could be useful for diagnosis of asymptomatic cases, as well as in the early phase of infections.

Evaluation of Cysteine Protease C of Leishmania donovani in Comparison with Glycoprotein 63 and Elongation Factor 1α for Diagnosis of Human Visceral Leishmaniasis and for Posttreatment Follow-Up Response.

Visceral leishmaniasis (VL) is a threat in many developing countries. Much effort has been put to eliminating this disease, for which serodiagnosis remains the mainstay for VL control programs. New and improved antigens as diagnostic candidates are required, though, as the available antigens fail to demonstrate equal optimum performance in all areas of endemicity. Moreover, these diagnoses are dependent on invasive serum sampling. In the current study, we cloned and expressed Leishmania donovani cysteine protease C (CPC) and evaluated its diagnostic and test-of-cure possibilities by detecting the antibody levels in human serum and urine through ELISA and immunoblot assays. Two immunodominant antigens, recombinant glycoprotein 63 (GP63) and elongation factor 1α (EF1α), identified earlier by our group, were also assessed by employing human serum and urine samples. Of these three antigens in ELISAs, CPC demonstrated the highest sensitivities of 98.15% and 96% positive testing in serum and urine of VL patients, respectively. Moreover, CPC yielded 100% specificity with serum and urine of nonendemic healthy controls compared to GP63 and EF1α. Urine samples were found to be more specific than serum for distinguishing endemic healthy controls and other diseases by means of all three antigens. In all cases, CPC gave the most promising results. Unlike serum, urine tests demonstrated a significant decrease in antibody levels for CPC, GP63, and EF1α after 6 months of treatment. The diagnostic and test-of-cure performances of CPC in the immunoblot assay were found to be better than those of GP63 and EF1α. In conclusion, CPC, followed by GP63 and EF1α, may be utilized as candidates for diagnosis of VL and to assess treatment response.

Development and Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Diagnosis of Human Visceral Leishmaniasis in Brazil.

Visceral leishmaniasis (VL) is considered a major public health concern in Brazil and several regions of the world. A recent advance in the diagnosis of infectious diseases was the development of loop-mediated isothermal amplification (LAMP). The aim of this study was to develop and evaluate a new LAMP assay for detection of K26 antigen-coding gene of L. donovani complex. A total of 219 blood samples of immunocompetent patients, including 114 VL cases and 105 non-VL cases, were analyzed for the diagnosis of VL in the present study. Diagnostic accuracy was calculated against a combination of parasitological and/or serological tests as a reference standard. The results were compared to those of kDNA Leishmania-PCR. The detection limit for the K26-Lamp assay was 1fg L. infantum purified DNA and 100 parasites/mL within 60 min of amplification time with visual detection for turbidity. The assay was specific for L. donovani complex. Sensitivity, specificity, and accuracy were 98.2%, 98.1%, and 98.2%, respectively, for K26-LAMP and 100%, 100%, and 100%, respectively, for kDNA Leishmania-PCR. Excellent agreement was observed between K26-LAMP and kDNA Leishmania-PCR assays (K = 0.96). A highly sensitive and specific LAMP assay targeting K26 antigen-coding gene of L. donovani complex was developed for diagnosis in peripheral blood samples of VL patients.

Where, when, and how the diagnosis of human visceral leishmaniasis is defined: answers from the Brazilian control program.

BACKGROUND Timely diagnosis is recommended by the Brazilian Visceral Leishmaniasis (VL) Surveillance and Control Program to reduce case fatality. Attempts at assessing this topic in Brazil are scarce. OBJECTIVE This study aimed to describe where, when, and how the diagnosis of VL has been performed in a Brazilian endemic setting.METHODS Data of all autochthonous cases confirmed between 2011 and 2016 (N = 81) were recorded. The care-seeking itinerary until the confirmation of VL diagnosis was assessed among 57 patients.FINDINGS The majority of VL cases (79.1%) were reported by referral hospitals. The patients mainly sought primary health care centres at the onset of symptoms. However, they had to visit seven health services on average to achieve a confirmed diagnosis. The time from the onset of symptoms to the diagnosis of VL (TD) ranged from 1-212 (median, 25) days. The TD was longer among adult patients. There was a direct correlation between the patient’s age and TD (r = 0.22; p = 0.047) and a higher occurrence of deaths due to the disease among older patients (p = 0.002). Almost all the patients (98.9%) underwent laboratory investigation, and the VL diagnosis was mainly confirmed based on clinical-laboratory criteria (92.6%). Positive results for the indirect fluorescence antibody test (22.7%) and parasitological examination plus rk39-based immunochromatographic tests (21.3%) were commonly employed.MAIN CONCLUSIONS VL diagnosis was predominantly conducted in hospitals with a long TD and wide application of serology. These findings may support measures focused on early diagnosis, including a greater involvement of the primary health care system.

New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening.

Visceral leishmaniasis (VL) still represents a serious public health problem in Brazil due to the inefficiency of the control measures currently employed, that included early diagnosis and treatment of human cases, vector control, euthanasia of infected dogs and, recently approved in Brazil, treatment with Milteforam drug. Effective clinical management depend largely on early and unequivocal diagnosis, however, cross-reactivity have also been described in serological tests, especially when it refers to individuals from areas where Chagas’ disease is also present. Thus, to discover new antigens to improve the current serological tests for VL diagnosis is urgently needed. Here, we performed an immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted L. infantum proteome using the following criteria: i) proteins expressed in the stages found in the vertebrate host, amastigote stage, and secreted/excreted, to guarantee greater exposure to the immune system; ii) divergent from proteins present in other infectious disease pathogens with incidence in endemic areas for VL, as T. cruzi; iii) highly antigenic to humans with different genetic backgrounds, independently of the clinical stage of the disease; iv) stable and adaptable to quality-control tests to guarantee reproducibility; v) using statistical analysis to determine a suitable sample size to evaluate accuracy of diagnostic tests established by receiver operating characteristic strategy. We selected six predicted linear B-cell epitopes from three proteins of L. infantum parasite. The results demonstrated that a mixture of peptides (Mix IV: peptides 3+6) were able to identify VL cases and simultaneously able to discriminate infections caused by T. cruzi parasite with high accuracy (100.00%) and perfect agreement (Kappa index = 1.000) with direct methods performed by laboratories in Brazil. The results also demonstrated that peptide-6, Mix III (peptides 2+6) and I (peptides 2+3+6) are potential antigens able to used in VL diagnosis, represented by high accuracy (Ac = 99.52%, 99.52% and 98.56%, respectively). This study represents an interesting strategy for discovery new antigens applied to serologic diagnosis which will contribute to the improvement of the diagnosis of VL and, consequently, may help in the prevention, control and treatment of the disease in endemic areas of Brazil.

Development and Validation of a PCR-ELISA for the Diagnosis of Symptomatic and Asymptomatic Infection by Leishmania (Leishmania) infantum.

A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the kDNA PCR-ELISA were defined in a proof-of-concept phase. A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with Leishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The kDNA PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from L. (L.) infantum and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1–100%), and the specificity was 95% (95% CI: 83.5–98.6%). The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions.

Diagnostic accuracy of rKLO8 versus rK26 ELISAs for screening of canine visceral leishmaniasis

Canine visceral leishmaniasis (CVL) represents an important public health issue. Despite numerous diagnostic tests available, CVL diagnosis still needs to be improved to achieve a more accurate detection rate. Recently, rKLO8, a new antigenic protein of Sudanese Leishmania donovani, was studied for the first time in diagnosis of human visceral leishmaniasis (HVL) and showed good performance. The present study aimed to evaluate serum reactivity to rKL08 and the reference antigen rK26, and to compare both diagnostic proteins with the combined DPP® CVL rapid test and ELISA (EIE-Bio-Manguinhos) confirmatory test, which are both recommended for the diagnosis of CVL in Brazil. Serum samples of dogs were grouped into: (I) DPP®/EIE negative (n=100) and (II) DPP®/EIE positive sera (n=100). Enhanced levels of IgG, mainly IgG2, to both rKLO8 and rK26 were found in group II. Sensitivity was 68% and 77% and specificity was 92% and 91%, for rKLO8 and rK26 antigens, respectively. Moreover, the combination of rKLO8 and rK26 antigens (rKLO8+rK26) exhibited higher sensitivity (85%) and specificity (93%). Thus, our results show that apart from the improved diagnostic power of rKLO8 in HVL, this new antigen is also suitable for the diagnosis of CVL. Further, the combination of rKLO8 and rK26 antigens increases the diagnostic accuracy of CVL.

Laboratory diagnosis of human visceral leishmaniasis

Laboratory diagnosis of human visceral leishmaniasis

Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

ObjectiveTo evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. MethodsDNA extraction was performed using Promega Wizard® Genomic kits. PCR employing RV1/RV2 primers yielded 145-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. ResultsThe sensitivity and specificity levels were 91,3%% and 29,6%, respectively, for real-time PCR and 97.78% and 61.82%, respectively, for PCR. ConclusionsReal-time PCR proved to be a satisfactory method for the diagnosis of human visceral leishmaniasis.

Diagnosis of Human Visceral Leishmaniasis by PCR Technique in Iraq

In the present study, 93 blood samples and 20 bone marrow aspirates from 93 patients with clinical symptoms suggestive of visceral leishmaniasis (VL) from three hospitals in Baghdad during the period October 2005 - May 2006, were screened using molecular and serological techniques. Samples that were negative (N = 50) in parasitological and serological tests were used as controls. Of the 93 samples analyzed by ELISA technique, 19 samples (17.7%) gave positive culture result on NNN medium. However, when the technique was combined with molecular characterization by PCR, 7 samples (36.8%) gave a positive result. Nineteen patients with Leishmania parasites in bone marrow cultures showed PCR amplification of the correct band, as did the seven bone marrow aspirates positive for Leishmania. None of the negative controls tested using PCR or in an ELISA assay. These results indicate that PCR could replace the conventional parasitological examination in the diagnosis of leishmaniasis since it provides very satisfactory results with clinical samples

Pediatric visceral leishmaniasis diagnosis in Tunisia: comparative study between optimised PCR assays and parasitological methods

There has been a steady increase of visceral leishmaniasis during the past 20 years in Tunisia. In this study, we assess the value of two optimised PCR versus those of classical methods for the diagnosis of human visceral leishmaniasis. 106 samples were collected from 53 cases of pediatric visceral leishmaniasis. Peripheral blood and bone marrow samples were analysed both by parasitological methods (direct examination, leukocytoconcentration (LCC) and culture) and by PCR methods with two primer pair (R221/R332 and Lei 70L/Lei 70R). We diagnosed visceral leishmaniasis in all patients: 44 cases were diagnosed by culture (83%), 42 by direct examination of bone marrow (79%), 17 by LCC (32%), and 53 positive cases with both PCR assays (R221/R332 and/or Lei 70L/Lei 70R) (100 %). Regarding each PCR assay, for blood samples, the difference between the sensitivities of PCR Lei 70L/Lei 70R (86,8%) and PCR R221/R332 (17 %) is statistically significant with p-value 0.025. For bone marrow, the sensitivities of the two PCR methods were respectively 96,2% (Lei 70L/Lei 70R) and 75,5% (R221/R332). On the whole, PCR Lei 70L/Lei 70R was more effective than PCR R221/R332 and conventional methods for the two biological samples. Moreover, the requirement of less invasive sample using blood has the advantage of being repeatable for screening and for post therapeutic monitoring.

Leishmania infantum DNA Detection in Urine from Patients with Visceral Leishmaniasis and after Treatment Control

A urine-polymerase chain reaction (PCR) assay was validated for diagnosis of human visceral leishmaniasis (VL), taking advantage of the accessibility of urine samples. Leishmania infantum DNA presence was examined in 17 urine samples from 17 patients with VL during a clinical episode and in 55 urine samples from 17 patients with VL monitored after treatment at different intervals. Fifty-nine urine samples from 59 controls with no history of VL were also studied. The urine-PCR test was positive in 15/17 samples obtained during the episode (sensitivity, 88%). None of the controls tested were urine-PCR positive (specificity, 100%). During the monitoring period, 25% of the samples gave a positive urine-PCR. Results were compared with other diagnostic methods, such as urine antigen detection and peripheral blood-PCR and culture, with good concordance during the clinical episode and differences in the follow-up period. This study suggests that urine-PCR is sensitive for diagnosis and may be useful to monitor treatment efficacy.

A Novel Exo-antigen-based ELISA for the Detection of Canine Leishmaniasis

Dogs which are infected with leishmania parasites serve as major reservoir hosts for zoonotic visceral leishmaniasis. The incidence of zoonotic visceral leishmaniasis is rising in many countries. This may be associated with the continuing drift of people and their pets from rural areas into peri-urban settings, particularly at the fringe of large cities. At the same time, there is evidence of adaptation of sand fly vectors to these urban settings. This has created an alarming situation because, even though domestic and stray dogs may be infected, many remain asymptomatic but are still highly infectious to the sand fly vectors and thus pose a serious threat to human health. Over half of the infected dogs have asymptomatic infections and current assays are not sensitive enough under field conditions to distinguish asymptomatic from symptomatic dogs. There is an urgent need for a specific and sensitive screening tool for use in the field. We have previously demonstrated that promastigote exo-antigen-based ELISAs can be used in the specific diagnosis of human visceral leishmaniasis (HVL). A cocktail of exo-antigens prepared from three species (L. infantum, L. donovani, and L. major) was used to develop and optimize a canine ELISA assay. Serum samples from dogs with a variety of pathological conditions but living in a non-leishmania endemic area were used as negative controls and their reactivity was used to determine a cut-off value for the ELISA. Samples from dogs residing in a leishmania endemic area were tested in parallel using direct agglutination (DAT), immunofluorescence (IFAT), and ELISA. The ELISA results correlated closely (100%) with the clinical symptoms, and were elevated in one asymptomatic dog. This sample was also found to be positive by IFAT. Based on its sensitivity and specificity, the cocktail exo-antigen-based ELISA may prove useful, even at 1:2,000 serum dilutions, for screening dogs in different geographical regions of the world.

Evaluación de una prueba de aglutinación directa como método alternativo para el diagnóstico de leishmaniasis visceral canina y humana en Venezuela

Visceral leishmaniasis is the most severe clinical form of leishmaniasis and is often fatal without proper treatment. Therefore, early and accurate diagnosis is important, but often difficult in endemic areas. The aim was to evaluate a direct agglutination test as a potential visceral leishmaniasis diagnostic method in endemic areas of Venezuela. The performance of the direct agglutination test, based on freeze-dried Leishmania donovani antigen was evaluated under laboratory conditions using serum samples of humans and dogs from several Venezuelan visceral leishmaniasis endemic areas: Nueva Esparta (Margarita Island), Lara, Anzoátegui and Trujillo Status. The study included confirmed visceral leishmaniasis patients (n=30), visceral leishmaniasis suspected subjects (n=4), healthy controls (n=19) and patients with other confirmed diseases (n=20). In addition, 24 serum samples from dogs with confirmed visceral leishmaniasis and 18 healthy control dogs were tested. All serum samples of visceral leishmaniasis patients, either active or recovered, were positive. They showed anti-L. donovani titers above 1:1600. Three out of four suspected visceral leishmaniasis cases were also positive, while serum samples from endemic controls and patients with other diseases had titers lower than 1:800. A sensitivity of 100% was obtained for all threshold levels under consideration and 100% specificity at the threshold titer of 1:800 (95% confidence interval: 91-100%). A 93% sensitivity (95% confidence interval: 76-99%) was observed in dog samples, with 100% specificity (95% confidence interval: 79-100%) at the threshold titer of 1:200. The direct agglutination test seems suitable for use in epidemiological studies and for serological diagnosis of human visceral leishmaniasis and canine visceral leishmaniasis.

Detection of circulating Leishmania chagasi DNA for the non-invasive diagnosis of human infection

A polymerase chain reaction (PCR) assay for the detection of Leishmania spp. DNA in peripheral blood was optimized and evaluated for the diagnosis of human visceral leishmaniasis (VL) in Brazil during May 2001 to December 2002. Optimization of the technique resulted in a detection limit of 1.65 fg of purified L. (L.) chagasi DNA, equivalent to 1.65 × 10 −2 parasites. Leishmania DNA was detected in the blood of 48 of 53 patients with parasitologically-confirmed VL, which corresponds to a sensitivity of 91%. No DNA was detected in the peripheral blood of 15 healthy, non-exposed volunteers, giving a specificity of 100%. We conclude that detection of parasite DNA in peripheral blood offers a non-invasive, sensitive and rapid method for the detection of VL caused by L. (L.) chagasi.