Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis

This study evaluated the performance of recombinant K39 (rK39) antigen in a immunochromatographic format (strip test) and a crude antigen enzyme-linked immunosorbent assay in the diagnosis of Brazilian visceral leishmaniasis (VL) in 128 consecutive patients with parasitologically proven infections (by microscopy and/or culture). For each patient, a medical history was obtained and a complete physical examination was performed. Controls included 10 healthy volunteers and 50 patients with other diseases: malaria (10), leprosy (9), Chagas' disease (10), tuberculosis (10), and cutaneous leishmaniasis (11). The sensitivities of the rK39 antigen strip test and the ELISA were 90% and 89%, respectively, while the specificities were 100% and 98%, respectively. Our study confirms the accuracy of the rK39 antigen strip test in the diagnosis of VL in a high prevalence population.     

A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi suitable for serodiagnosis of American visceral leishmaniasis

A recombinant protein, rLdccys1, which was produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used for detection of antibodies in sera from patients with active visceral leishmaniasis (VL) in enzyme-linked immunosorbent assays. Analysis of the predicted amino acid sequence of rLdccys1 showed that it contains all the characteristics of a cysteine proteinase. The ability of the protein to react with sera from humans with VL was also shown by Western blotting. The sensitivity for detection of specific antibodies to L. (L.) chagasi bodies using rLdccys1, L. (L.) chagasi promastigote lysates, and amastigote lysates was 80%, 98%, and 99%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and there was little positive reactivity with sera from patients with cutaneous leishmaniasis and tuberculosis, compared with promastigote and amastigote extracts. Our findings indicate that rLdccys1 from L. (L.) chagasi constitutes a potential tool for the diagnosis of American VL.     

Leishmania chagasi antigens recognized in cured visceral leishmaniasis and asymptomatic infection.

Active visceral leishmaniasis is associated with antigen-specific immuno-suppression. However, cured patients develop a cellular immune response associated with resistance to reinfection. Recent studies have identified patients with asymptomatic or subclinical infections, which are also accompanied by an immune response. In order to identify subjects immune to Leishmania chagasi, we performed a skin-test survey in an endemic area in eastern Venezuela. The delayed-type hypersensitivity (DTH) response was assessed in patients cured of visceral leishmaniasis, as well as in their relatives and neighbors. Of the latter, 36 (34.2%) of 105 were positive and 26 (24.7%) of 105 gave intermediate responses. The DTH reaction correlated with age. The antigens recognized by a subgroup of cured patients, those with positive skin-test results, and controls (skin-test negative) were assessed by Western blotting with sera, and T cell immunoblotting with peripheral blood mononuclear cells. No consistent differences between the groups were noted in Western blots with L. chagasi antigens. T cell blots were performed on five patients from each group. For the cured patients and skin-test positive contacts, a significant proliferative response to fraction 12 (less than 20.5 kDa) was noted in four of five patients in each group. Cells from three of five cured patients and two of five skin-test-positive patients proliferated in response to fraction 4 (73-115 kDa). The response to other fractions was variable, with only a minority of patients responding to any one fraction. These data suggest that the antigens recognized by patients with evidence of immunity to L. chagasi are quite variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

The burden of Leishmania chagasi infection during an urban outbreak of visceral leishmaniasis in Brazil

First noted in the city of Teresina in 1981, the last decades have witnessed a remarkable increase in urban transmission of American visceral leishmaniasis (VL) in many Brazilian cities. Teresina, the site of this study, has faced two large outbreaks of VL. The first occurred from 1981-1985 when almost 1000 new cases were reported. The second started in the 1990s, and between 1993 and 1996 more than 1200 new cases were detected. This report describes the prevalence of infection with Leishmania chagasi in Teresina at the end of the second outbreak and gives estimates of the number of people who became infected during the epidemic. Between June 1995 and May 1996, 200 households were chosen at random from a list of addresses covering about 93% of Teresina's urban households. In each household, one person over the age of 1 year was screened for Leishmania antibodies and skin-tested. Nearly 50% of persons had a positive leishmanin reaction, but only 13.9% had detectable antibodies to L. chagasi. While prevalence estimates based on the leishmanin skin-test increased with age (P<0.001), those based on serological tests showed a lesser, and non significant, variation with age (P=0.31). Using a geometric growth equation, and assuming that the annual distribution of clinical cases may serve as an approximation to what would have been the distribution of infections by year, we estimated that over 320000 persons were infected during the epidemic. Little is known about the epidemiology of VL in urban areas, where social networks, population density, and relationships of housing with the natural environment are more varied and complex than in the rural scene. In those areas, control interventions have failed to eliminate transmission of the parasite and prevent new epidemics. Further epidemiological studies of VL in urban areas might be needed to inform control actions.     

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.     

Immunofluorescent antibody test in American visceral leishmaniasis: sensitivity and specificity of different morphological forms of two Leishmania species

This study was designed to determine which morphologic form and species of Leishmania is most suitable for detection of antibody in sera from American visceral leishmaniasis (AVL) patients by the indirect fluorescent antibody test (IFAT). Promastigotes and amastigotes of Leishmania mexicana or Leishmania donovani chagasi were used as sources of antigen. A total of 70 sera, including 30 from AVL patients, 30 from healthy subjects and 10 from Chagas' disease patients, were used in the study. Titers of antibody up to a dilution of 1:64 were found with all four antigens. At a titer of 1:128, the sensitivity of the IFAT using L. d. chagasi promastigotes as a source of antigen was 100% and the specificity at a titer of 1:32 was 98%. Although the sensitivity of the amastigote forms was close to 100% at a similar titer, the specificity at a titer of 32 using the L. d. chagasi amastigotes was 91% and using L. mexicana amastigotes was only 80%. The L. d. chagasi promastigote antigen was also the one that showed less cross reaction with sera from Chagas' disease patients. Since cross reactivity between Trypanosoma cruzi and Leishmania species is well known in serological tests, and minimizing of such cross reactivity is of critical importance for diagnosis, we suggest that L. d. chagasi promastigotes should be the antigen of choice for diagnosis of visceral leishmaniasis by IFAT in areas also endemic for trypanosomiasis.     

Human visceral leishmaniasis: analysis of the specificity of humoral immune response to polypeptides of Leishmania donovani chagasi

Soluble antigens from Leishmania donovani chagasi were studied in terms of their ability to react with sera from human visceral leishmaniasis. Thirty-six polypeptides, with molecular weights ranging from 14,400 to 123,000 were demonstrated by Western blot analysis. An extensive cross-reactivity with sera from patients with cutaneous leishmaniasis and Chagas' disease also was observed. Two polypeptides (Mr 119,000 and 123,000) reacted with all the sera from visceral leishmaniasis patients. When they were electroeluted from gels and evaluated with respect to specificity to the L. donovani chagasi subspecies, these components were expressed in all strains of Leishmania tested, but not in those of Trypanosoma cruzi. These results indicated that these components are shared by Trypanosomatidae of genus Leishmania. The eluted polypeptides did not react with sera from patients with Chagas' disease, indicating the feasibility of using purified antigens to discriminate between the humoral immune responses in T. cruzi and Leishmania infections.     

An emerging peri-urban pattern of infection with Leishmania chagasi, the protozoan causing visceral leishmaniasis in northeast Brazil

Peri-urban visceral leishmaniasis (VL) caused by Leishmania chagasi is emerging in a new epidemiologic pattern in Brazilian cities. We studied peri-urban VL in endemic neighborhoods surrounding Natal, Brazil, identified through hospitalized individuals with VL. Clinical and environmental information obtained for 1106 members of 216 families living in endemic neighborhoods enabled us to identify 4 groups: VL: individuals with current or prior symptomatic visceral leishmaniasis (n = 135); DTH+: individuals with positive delayed-type hypersensitivity response with no history of VL (n = 390); Ab +: individuals with negative DTH response and seropositive (n = 21); DTH -: individuals with negative DTH and seronegative (n = 560). The mean +/-SD age of VL was 9.3+/-12.3 y. The gender distribution was nearly equal below age 5, but skewed toward males at higher ages. Acutely infected VL subjects had significantly lower hematocrits, neutrophils, and eosinophils than other categories. AB+ subjects also had lower eosinophil counts than others, a possible immune marker of early infection. VL was not associated with ownership of dogs or other animals, raising the question whether the reservoir differs in peri-urban settings. This new pattern of L. chagasi infection enables us to identify epidemiological and host factors underlying this emerging infectious disease.     

Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan

We compared a strip test employing recombinant K39 (rK39) antigen and protein A/colloidal gold as read-out agents with the rK39 ELISA for IgM and IgG antibodies and the direct agglutination test (DAT) using 55 sera from patients with parasitologically confirmed visceral leishmaniasis (VL). The rK39 strip test was positive in 37/55 (67%), the DAT in 50/55 (91%) at > or = 1 : 1600 cut-off value and in 47/55 (85%) at > or = 1 : 6400 cut-off value. The rK39-ELISA gave positive IgG results for all sera; those who had a positive strip test had significantly higher IgG levels than those with a negative strip test (31.1 (SD=3.6) and 17.7 U/ml (SD=9.8), respectively, P < 0.0001). A total of 31/55 (56%) sera showed a positive IgM result; of these 27 (49%) had a positive strip test. We tested 115 apparently cured VL patients with the strip test during follow-up; 68 were also tested with DAT. In the strip test, 25-43% of patients had a positive result at time points 3, 6, 9 and 12 months after treatment; for DAT (cut-off > or = 1 : 1600) these results were 67-83%. In neither test did a significant decrease in positivity rates occur over time (P=0.37 for the strip test, P=0.17 for the DAT). No correlation (P=0.33) was found between a positive strip test and a positive DAT result (cut-off > or = 1: 1600), indicating that the strip test and DAT are complementary rather than interchangeable. Of 61 endemic controls two (3%) had a positive strip test result; both had a positive leishmanin skin test. The rK39 strip test has the ideal format for use in the field, but its sensitivity is limited; like DAT, but to a lesser extent, it remains positive after treatment.     

The high sensitivity of a PCR-ELISA in the diagnosis of cutaneous and visceral leishmaniasis caused by Leishmania infantum

In general, the conventional techniques available for the diagnosis of leishmaniasis have relatively low sensitivity. This means that parasite-rich samples (which can usually only be collected by very invasive methods, such as bone-marrow aspiration) must be employed. This problem has not yet been solved even by use of the PCR-based techniques currently available. However, a new PCR-ELISA has been developed for the diagnosis of cutaneous (CL) and visceral leishmaniasis (VL) caused by Leishmania infantum. This assay appears to have sufficient sensitivity to be effective in the diagnosis of VL not only when bone-marrow aspirates are investigated but also when the samples are of peripheral blood. Overall, the ability of the PCR-ELISA to detect Leishmania, in 76 samples (22 of peripheral blood, 36 bone-marrow aspirates and 18 skin samples) from 72 patients living in a endemic region, was better than that of culture or the examination of Giemsa-stained smears. For example, L. infantum kDNA was detected by PCR-ELISA in 15 (83%) of the 18 skin samples from suspected cases of CL, whereas the combined use of several classical techniques only confirmed the presence of amastigotes in five (28%) of these samples. Similarly, only 21 individuals were diagnosed as having VL by conventional techniques whereas 30 were found Leishmania-positive in the PCR-ELISA. The new PCR-ELISA also appears to be a suitable technique for detecting leishmanial kDNA in samples of peripheral blood from cases of L. infantum-HIV co-infection. The assay is more sensitive than the combined use of several conventional techniques in the diagnosis of subclinical VL, probably because those with subclinical infection have relatively low parasitic loads that are generally undetectable using the other techniques.     

rK39 antigen for the diagnosis of visceral leishmaniasis by using human saliva

The rK39 rapid immunochromatographic test (ICT) is now being widely used in the diagnosis of visceral leishmaniasis (VL) using serum. We evaluated the presence of anti-rK-39 antibody in human saliva being noninvasive to replace the invasive procedures of diagnosis. Enzyme-linked immunosorbent assay (ELISA) and ICT assays were performed in 300 subjects: 114-confirmed VL patients, 95 and 47 healthy controls from endemic and nonendemic regions, respectively, and 44 subjects with different diseases. Sensitivity in saliva was 83.3% by ELISA and 82.5% by ICT, compared with 100% for both ICT and ELISA in serum. Specificity in saliva was 100%, 90.5%, and 88.6% with ELISA, and 91.48%, 91.57%, and 84.06% using ICT, in nonendemic, endemic, and different diseases, respectively. In serum, specificity was 97%, 88.5%, and 89% by ELISA and 100%, 94.7%, and 95.5% by ICT in nonendemic, endemic, and different diseases, respectively. Saliva is not suitable for diagnosis of VL because of low sensitivity.     

The burden of the Leishmania chagasi/infantum infection in a closed rural focus of visceral leishmaniasis in Lara state, west-central Venezuela

A prospective study was conducted in El Brazilar, Curarigua, Lara State, Venezuela, a small rural focus of visceral leishmaniasis (VL) to investigate the burden and the evolution of Leishmania infection in the human and canine population. The incidence of the disease from February 1998 to February 2002 was recorded and two cross-sectional surveys using the leishmanin skin test (LST) and immunofluorescent antibody test (IFAT) were carried out. The dipstick test with recombinant r-K39 antigen was also applied in 2002. The incidence of the disease per year among the population (n=118) during the period of study was 0.004. The rate of new infections per year was 0.07 [95% confidence interval (95% CI): 0.15-1.09]. The overall prevalence of infection measured by LST was not significantly higher in 2002 (43.2%) than in 1998 (28.3%), but it was with IFAT [16.3%vs. 4.6%; odds ratio (OR): 4.01; 95% CI: 1.03-22.78; P=0.022] which would indicate an increasing transmission. The dipstick test only detected infection in children up to 10 years (19.4%). Prevalence in dogs was not significantly different in 2002 (57%) vs. 1998 (33%). The parasite was isolated from dogs and identified by a polymerase chain reaction based on telomeric sequences as Leishmania chagasi/infantum.     

Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral leishmaniasis

A new rK39 rapid diagnostic dipstick test (DiaMed-IT-Leish) was compared with aspiration and a direct agglutination test (DAT) for diagnosis of visceral leishmaniasis (VL) in 201 parasitologically confirmed cases, 133 endemic controls, and in 356 clinical suspects in disease-endemic and -epidemic areas in Sudan. The sensitivity of the rK39 test in parasitologically confirmed VL cases was 90%, whereas the specificity in disease-endemic controls was 99%. The sensitivity of the DAT was 98%. In clinically suspected cases, the sensitivity of the rK39 test was 81% and the specificity was 97%. When compared with the diagnostic protocol based on the DAT and aspiration used by Médecins sans Frontières in epidemic situations, the positive predictive value was 98%, and the negative predictive value was 71%. This rK39 rapid diagnostic test is suitable for screening as well as diagnosis of VL. Further diagnostic work-up of dipstick-negative patients with clinically suspected VL is important. The ease and convenience of the dipstick test will allow decentralization and improved access to care in disease-endemic areas in Sudan.     

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP‐HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients’ urine. Specifically, these proteins were the following: Li‐isd1 ( XP_001467866.1 ), Li‐txn1 ( XP_001466642.1 ) and Li‐ntf2 ( XP_001463738.1 ). Initial vaccine validation studies were performed with the rLi‐ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA‐SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li‐ntf2+ BpMPLA‐SE had a marked parasite burden reduction in spleens at 40 days post‐challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.     

Immunogenicity and efficacy of recombinant 78 kDa antigen of Leishmania donovani formulated in various adjuvants against murine visceral leishmaniasis

Objective: To analyze the protective efficacy of recombinant 78 kDa antigen of Leishmania donovani in combination with two adjuvants, that is, cationic liposomes or MPL-A against visceral leishmaniasis in BALB/c mice. Methods: The genomic DNA of promastigotes was isolated and 583 bp of T cell epitopes of gene encoding 78 k Da was amplified using specific primers. The amplified gene was cloned into p ET28 c, transformed into Escherichia coli BL21(DE3) and got expressed after IPTG induction. The recombinant protein was then purified using Ni-NTA and named r78. Three groups of mice were immunized with 10 μg of r78 plus MPL-A, r78 encapsulated in positively charged liposomes and control animals immunized with PBS. Two booster doses were given with the respective vaccine at an interval of 2 weeks each. Mice were challenged with 1×107 Leishmania promastigotes and sacrificed on different post infection/challenge days. Results: Immunization with r78 along with MPL-A and liposomeencapsulated r78 brought a significant reduction in parasite load. In comparison to the infected controls, the parasite load declined by 96.2% in mice immunized with r78 plus MPL-A and 97.23% in animals immunized with liposome-encapsulated r78. The immunized animals also exhibited profound DTH response. The serum antibody responses increased from 15 to 90 days post infection/challenge. Immunized animals showed greater IgG2 a levels and lesser Ig G1 levels in comparison to the infected controls. The splenocytes from immunized mice were cultured, stimulated with r78 and analyzed for cytokine profile. The levels of IL-2 and IFN-γ were greater in immunized animals as compared to control mice. Conclusions: The study proves that r78 in combination with suitable adjuvants is a potential vaccine candidate and may be instrumental in control of visceral leishmaniasis.     

Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis

Canine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leishmaniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI = 92.60–100.0%) and a high specificity of 100% (95% CI = 86.77–100.0%), with a high degree of confidence (k = 1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis.     

Isolation of Leishmania mexicana amazonensis from the bone marrow in a case of American visceral leishmaniasis

The first documented human case of visceral leishmaniasis caused by L. mexicana amazonensis is reported. Leishmania were isolated from bone marrow aspirate material from a typical visceral leishmaniasis patient. Further characterization by isoenzyme electrophoresis and by a panel of species- and subspecies-specific monoclonal antibodies established its classification as L. m. amazonensis.     

Organ-specific immunity in canine visceral leishmaniasis: analysis of symptomatic and asymptomatic dogs naturally infected with Leishmania chagasi

We characterized key leukocyte immunophenotypes in the liver and spleen of naturally infected dogs from an area in Venezuela endemic for leishmaniasis. Dogs were classified as symptomatic or asymptomatic after serologic and physical analysis. Symptomatic dogs showed a higher parasite burden in the liver and spleen than asymptomatic dogs. The livers of asymptomatic dogs showed an effective immunity with well-organized granulomas walling off parasites in an environment of central memory CD44(lo), CD45RO(hi), activated effector CD44(hi), and CD45RO(hi) T cells. These granulomas also had many major histocompatibility class II+ cells and CD11c+ dendritic cells, and cells expressing CD18 and CD44. In contrast, symptomatic livers showed a non-organized and non-effective infiltrate composed of T cells and heavily parasitized Kupffer cells and a diminished expression of activation molecules. In the spleen, the immune responses of symptomatic and asymptomatic dogs were very similar. The results showed a distinct immune response against Leishmania chagasi in target organs.     

Sensitivity and specificity of the Leishmania OligoC‐TesT and NASBA‐oligochromatography for diagnosis of visceral leishmaniasis in Kenya

Objective To estimate the sensitivity and specificity of the OligoC‐TesT and nucleic acid sequence‐based amplification coupled to oligochromatography (NASBA‐OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya. Methods Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays. Results The Leishmania OligoC‐TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90–98.8%) and a specificity of 88.8% (95% CI: 81–93.6%), while the sensitivity and specificity of the NASBA‐OC were 79.8% (95% CI: 67–87%) and 100% (95% CI: 96.3–100%), respectively. Conclusion Our findings indicate high sensitivity of the Leishmania OligoC‐TesT on blood while the NASBA‐OC is a better marker for active disease.     

Identification and characterization of a novel Leishmania donovani antigen for serodiagnosis of visceral leishmaniasis

Despite several drawbacks, rK39-based rapid immunochromatographic test is widely used for the diagnosis of visceral leishmaniasis (VL) in the Indian subcontinent. There is an urgent need to develop a better antigen. In this study we separated crude soluble antigens of Leishmania donovani by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hybridized with pool sera from pre- and post-treated VL patients, 6 months follow-up, endemic healthy (EHC), and nonendemic healthy controls (NEHC) by Western blotting. The sensitivity of enzyme-linked immunosorbent assay with identified protein was 95% (confidence interval [CI] = 89.6-98.01%), whereas the specificity for EHC, NEHC, and different disease groups were 96.3% (CI = 89.8-98.6%), 100% (CI = 95.8-100%), and 97.4% (CI = 91.02-99.3%), respectively. This specific antigen was subjected to two-dimensional gel electrophoresis and after tryptic digestion, antigen was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Further analysis showed that it is a member of the heat shock protein family of 70 kDa, designated as BHUP1, and has great potential in the diagnosis of VL.