The NF-κB transcription factor c-Rel controls host defense against Citrobacter rodentium.

Mice lacking CD4+ T cells or B cells are highly susceptible to Citrobacter rodentium infection. In this study, we show that the activity of the transcription factor c-Rel in lymphocytes is crucial for clearance of C. rodentium. Mice deficient for c-Rel fail to generate protective antibodies and to eradicate the pathogen.

A hyperactive mutant of interferon-regulatory factor 4.

We found that deletion of the final 30 amino acids of transcription factor IRF4's (interferon-regulatory factor) C-terminus creates hyperactive IRF4. When introduced into IRF4-deficient CD4+ or CD8+ T cells, more type 17 differentiation was found compared to WT IRF4. Interestingly, Th9 differentiation and Th2-linked IL-13 production were much less altered.

Leishmaniasis in Saudi Arabia: Current situation and future perspectives.

BACKGROUND AND OBJECTIVE: Leishmaniasis is endemic in Saudi Arabia with cases reported in many regions. This review refers to publications on leishmaniasis in Saudi Arabia and discusses issues related to parasite species, clinical manifestation and diagnosis. METHODS: This research was done at Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia by systematic literature search on PubMed and Google Scholar databases from 1989 to 2018. Selection criteria included original articles reporting on visceral leishmaniasis (VL) or cutaneous leishmaniasis (CL) in Saudi Arabia. RESULTS: The search identified 16 eligible articles, six for VL and 10 for CL. VL was reported in areas known to be non-endemic. Leishmania donovani was the main cause for human VL while Leishmania infantum seemed to cause the disease in animals. Dogs were considered the main reservoir hosts and black rats (Rattus rattus) were potential hosts. VL mainly affected infants and young children. It is important to note that VL diagnosis was based on either invasive parasite detection procedures or serologically using indirect hemagglutination test. CL represented the most frequent clinical form with the main endemic foci reported in the South-West and Eastern regions. CL appeared to have no demographic or socioeconomic restriction; it affected both rural and urban citizens, with the majority occurring among farmers. Travelling was recognized as an important risk factor. Leishmania tropica and Leishmania major were recognized as the main causes for CL. CONCLUSION: This report summarizes the potential risks for VL and CL in Saudi Arabia in areas known to be non-endemic. There are substantial gaps in knowledge and practices in regard to leishmaniasis in Saudi Arabia, highlighting the need for more research and medical surveillance targeting the disease in humans and animals.

Phenotypic detection of Extended Spectrum ß-Lactamases (ESBL) among gram negative uropathogens reveals highly susceptibility to imipenem.

OBJECTIVE: We aimed to determine antibiotic susceptibility patterns of ESBL- and non-ESBL bacteria isolated from pregnant women with UTI in antenatal wards in Khartoum State, Sudan. METHODS: This cross-sectional study was conducted during April-July 2016 at different hospitals in Khartoum State. Mid-stream urine samples were obtained from 150 hospitalized pregnant women and cultured on CLED (Cystine Lactose Electrolyte Deficient) agar. Microorganisms were identified using standard microbiological procedures. Isolated Gram-negative bacteria were tested for antibiotic susceptibility and ESBL screening using modified Kirby- Bauer method and Double Disc Synergy Test (DDST) respectively. RESULTS: Urine culture revealed positive results in 33/150 (22%) and the most prevalent isolates were Gram negative bacteria (18/33, 54.5%). Among gram-negative bacteria, isolates of E. coli were the most prevalent accounting 66.6% (12/18) followed by K. pneumoniae (4/18, 22.2%) and K. oxytoca (2/18, 11.1%). ESBL was detected in 8/18 (44.4%) of the Gram-negative isolates. Of note, imipenem was the most susceptible antibiotic for ESBL-producer and non-ESBL producer Gram negative isolates, accounting 100% susceptibility for both bacterial groups. Overall susceptibility rates were also high for ciprofloxacin (13/18, 72.2%). In other hand, co-trimoxazole and amoxicillin showed high resistance pattern for ESBL-producer and non-ESBL producer isolates; 27.8%, 44.4% and 38.9%, 38.9% susceptibility rates of co-trimoxazole and amoxicillin for ESBL-producer and non-ESBL producer isolates, respectively. CONCLUSIONS: Imipenem remains the most powerful option for ESBL- and non-ESBL bacteria causing UTIs in pregnant women. However, due to tremendous increase of antibiotic-resistant, antibiotic-susceptibility testing is recommended as a routine investigation for admitted pregnant women.

Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation.

Follicular T-helper (T(FH)) cells cooperate with GL7(+)CD95(+) germinal center (GC) B cells to induce antibody maturation. Herein, we identify the transcription factor IRF4 as a T-cell intrinsic precondition for T(FH) cell differentiation and GC formation. After immunization with protein or infection with the protozoon Leishmania major, draining lymph nodes (LNs) of IFN-regulatory factor-4 (Irf4(-/-)) mice lacked GCs and GC B cells despite developing normal initial hyperplasia. GCs were also absent in Peyer's patches of naive Irf4(-/-) mice. Accordingly, CD4(+) T cells within the LNs and Peyer's patches failed to express the T(FH) key transcription factor B-cell lymphoma-6 and other T(FH)-related molecules. During chronic leishmaniasis, the draining Irf4(-/-) LNs disappeared because of massive cell death. Adoptive transfer of WT CD4(+) T cells or few L. major primed WT T(FH) cells reconstituted GC formation, GC B-cell differentiation, and LN cell survival. In support of a T-cell intrinsic IRF4 activity, Irf4(-/-) T(FH) cell differentiation was not rescued by close neighborhood to transferred WT T(FH) cells. Together with its known B lineage-specific roles during plasma cell maturation and class switch, our study places IRF4 in the center of antibody production toward T-cell-dependent antigens.

Comparative Study of a Novel Lateral Flow Rapid Test with Conventional Serological Test Systems for the Diagnosis of Canine Leishmaniosis in Croatia and Brazil.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot®Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

Development of a Novel Enzyme-Linked Immunosorbent Assay and Lateral Flow Test System for Improved Serodiagnosis of Visceral Leishmaniasis in Different Areas of Endemicity.

Visceral leishmaniasis (VL) is caused by protozoan parasites of the Leishmania donovani complex and is one of the most prominent vector-borne infectious diseases with epidemic and mortality potential if not correctly diagnosed and treated. East African countries suffer from a very high incidence of VL, and although several diagnostic tests are available for VL, diagnosis continues to represent a big challenge in these countries due to the lack of sensitivity and specificity of current serological tools. Based on bioinformatic analysis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed. The diagnostic performance of rKLi8.3 was evaluated by enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) on a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other diseases, including tuberculosis, malaria, and trypanosomiasis. The diagnostic accuracy of rKLi8.3 was compared with rK39 and rKLO8 antigens. The VL-specific sensitivity of rK39, rKLO8, and rKLi8.3 ranged from 91.2% over 92.4% to 97.1% and specificity ranged from 93.6% over 97.6% to 99.2%, respectively. In India, all tests showed a comparable specificity of 90.9%, while the sensitivity ranged from 94.7% to 100% (rKLi8.3). In contrast to commercial serodiagnostic tests, rKLi8.3-based ELISA and LFT showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer improved VL serodiagnostic efficiency in East Africa and other areas of endemicity. IMPORTANCE Reliable and field suitable serodiagnosis of visceral leishmaniasis (VL) in East Africa has until now been a big challenge due to low sensitivity and cross-reactivity with other pathogens. To improve VL serodiagnosis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed and tested with a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other infectious diseases. Both prototype rKLi8.3-based enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer substantially increased diagnostic efficiency for VL in East Africa and other areas of endemicity, compared to currently commercially available serodiagnostic tests.

c-Rel promotes type 1 and type 17 immune responses during Leishmania major infection.

Recent studies demonstrated the crucial role of c-Rel in directing Treg lineage commitment and its involvement in T helper 1 (Th1) cell-mediated autoimmune inflammation. We thus wondered whether these opposite functions of c-Rel influence the course of antiparasitic immune responses against Leishmania major, an accepted model for the impact of T-cell subsets on disease outcome. Here we show that c-Rel-deficient (rel(-/-) ) mice infected with L. major displayed dramatically exacerbated leishmaniasis and enhanced parasite burdens. In contrast to WT mice, IFN-γ and IL-17 production in response to L. major antigens was severely impaired in rel(-/-) mice. Reconstitution of Rag1(-/-) T-cell deficient mice with rel(-/-) CD4(+) T cells followed by L. major infection demonstrated that c-Rel-deficient T cells mount normal Th1 responses and are able to contain the infection. Similarly, Th1 differentiation of naïve CD4(+) cells in vitro was normal. Notably, a selective defect in IL-12 and IL-23 production was observed in rel(-/-) DCs compared with their WT counterparts. In conclusion, our data suggest that the expression of c-Rel in myeloid cells is essential for clearance of L. major and that this c-Rel-mediated effect is dominant over the lack of Tregs.

Genetic Variants of RPL5 and RPL9 Genes among Saudi Patients Diagnosed with Thrombosis.

BACKGROUND: Thrombosis directly affects the quality of life with increased mortality. The RPL5 (L5) gene on intron 6 on chromosome 1p22, rs6604026 is associated with multiple sclerosis risk, whereas RPL9 (L9) on 8 exons on chromosome 4p14 has been documented so far as being an essential involvement in the proliferation of protein synthesized cells mostly by gene products. OBJECTIVE: The aim of this work was to assess genetic variants of RPL5 and RPL9 and thrombosis to characterize their role in the diagnosis of thrombosis among the Saudi population. METHODS: The cross-sectional study involved 100 Saudi patients diagnosed with thrombosis (arterial or venous) in 50 healthy individuals as controls in the same age and sex groups. Primers were designed RPL5 and RPL9 for molecular analysis. The Sanger System ABI-3730xL (Hong Kong) automatic sequencing was used for DNA sequencing. Statistical analysis was performed using the Prism 5 and SPSS version-21 programs. RESULTS: The male / female age ratio was 66.7 / 57.4, and the mean age was 61.2 years. Most of the patients were self-identifiable and without a previous history of thrombosis (61.0%). Most of the patients had just been diagnosed, that is, in the last five years (74.0%), about 43% of the patients underwent treatment using combination therapy (Aspirin and oral anticoagulants). New gene variants of RPL5 (5 SNPs) and RPL9 (9 SNPs) were detected in Saudi thrombotic patients. CONCLUSION: Mutations in RPL5 and RPL9 were reported in all thrombotic patients, represented by a new variant of the ribosomal protein gene and correlated with thrombosis in the Saudi population. These results may reflect an association between the ribosomal protein SNP gene and the incidence and progression of thrombosis in the Saudi population.

Identification of RPL5 gene variants and the risk of hepatic vein thrombosis in Saudi patients.

OBJECTIVES: To identify ribosome protein L5 gene variants and the risk of hepatic vein thrombosis in Saudi patients. METHODS: A case-control study was conducted during the period of May 2018 to September 2019. Sixty-five patient cases of hepatic vein thrombosis (HVT) were chosen, and 50 healthy individuals of the same ages and both gender were set as a control group. The genotype of the gene RPL5 was determined by PCR please provide abbreviation in full and capillary electrophoresis. Sanger sequencing for genetically screened variants was applied for the RPL5 gene. RESULTS: Alleles A at variant rs182018447 and T allele at variant rs559377519 were strongly corelated (p=0.009 and p=0.037, respectively) with the risk of HVT. The genotype frequencies of the RPL5 gene, the A/A genotypes at rs182018447 and T/T at rs559377519 were associated with HVT (p=0.000 and p=0.004; respectively) and an increase in risk for HVT among these patients. Please rephrase the highlighted text without using the word respectively. CONCLUSION: Our findings indicate that the 5 genetic novel variants examined in the RPL5 gene were associated with a risk of HVT in all our Saudi cases. Additionally, the A/A at rs182018447 and T/T at rs559377519 genotypes were substantially susceptible to HVT in all these patients.

Airborne transmission of SARS-CoV-2 is the dominant route of transmission: droplets and aerosols.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic worldwide. On a daily basis the number of deaths associated with COVID-19 is rapidly increasing. The main transmission route of SARS-CoV-2 is through the air (airborne transmission). This review details the airborne transmission of SARS-CoV-2, the aerodynamics, and different modes of transmission (e.g. droplets, droplet nuclei, and aerosol particles). SARS-CoV-2 can be transmitted by an infected person during activities such as expiration, coughing, sneezing, and talking. During such activities and some medical procedures, aerosols and droplets contaminated with SARS-CoV-2 particles are formed. Depending on their sizes and the environmental conditions, such particles stay viable in the air for varying time periods and can cause infection in a susceptible host. Very few studies have been conducted to establish the mechanism or the aerodynamics of virus-loaded particles and droplets in causing infection. In this review we discuss the various forms in which SARS-CoV-2 virus particles can be transmitted in air and cause infections.

Low serum zinc concentrations in Sudanese patients with visceral leishmaniosis does not impair the anti-Leishmania antibody response.

Visceral leishmaniosis (VL) is a poverty-related disease, affecting poor peoples in the developing countries. For unknown reasons, the rK39 rapid test has poor diagnostic sensitivity in some endemic areas such as East Africa. Here, the hypothesis was tested whether micronutrient deficiency is associated with low Leishmania-specific antibody responses and consequently decreased diagnostic sensitivity. Serum zinc concentrations in 107 human sera of VL and controls that were HIV-negative were measured using atomic absorption spectroscopy. Anti-Leishmania donovani antibodies were detected quantitatively by rKLO8 ELISA. The influence of low serum zinc concentrations on the amount of anti-Leishmania antibody and outcome of the rK39 rapid test was tested. Serum zinc concentrations were significantly (p ˂ 0.0001) reduced in VL sera (mean 0.41 ± 0.15) as compared to healthy control groups (mean ˃ 0.75 ± 0.13). Interestingly, the majority (92.2%) of the VL patients had low serum zinc concentrations (˂ 0.6 mg/l) whereas all healthy controls showed normal levels. Unexpectedly, VL sera with normal (0.94-0.6 mg/l) or low (˂ 0.6 mg/l) zinc concentrations demonstrated no significant difference in amounts of Leishmania antibodies. In addition, VL sera with positive or negative rK39 rapid test results demonstrated similar serum zinc concentrations; mean values of 0.39 ± 0.14 mg/l and 0.38 ± 0.1 mg/l for VL sera of positive or negative rK39 results, respectively. Low serum zinc concentration seems not to play an important role in lowering anti-L. donovani antibody titers observed among Sudanese VL patients and doesn't affect rK39 rapid test results.

Serological screening of HIV and viral hepatitis revealed low prevalence among visceral leishmaniosis patients in Sudan.

There is an increasing concern about the co-infection of visceral leishmaniosis (VL) with human immunodeficiency virus (HIV) and/or viral hepatitis B/C. The aim of this study was to determine the prevalence of HIV and viral hepatitis co-infections among VL patients in a hyperendemic area in Eastern Sudan and to assess antibody levels in co-infected patients. This is a retrospective study where the sera of confirmed VL cases and non-VL individuals were analysed. The sera were screened for co-infections using immunochromatographic tests and ELISA for anti-HIV 1+2 antibodies, hepatitis B surface antigen (HBsAg), and anti-hepatitis C virus (HCV). Anti-Leishmania donovani antibodies in the sera of VL alone were assessed and compared to the sera of co-infected patients. Of the 100 screened VL sera, 6 (6%), 0 (0%), and 1 (1%) were positive for HBsAg, anti-HCV, and anti-HIV, respectively. These values were 5 (5%), 0 (0%), and 1 (1%) in the control group. Of note, the HCV screening test (Biorex, UK) showed positive reactivity in 32 (32%) and 17 (17%) sera of VL and control groups, respectively. All reactive sera tested negative in HCV ELISA. Of the 93 VL sera, 75 (80.6%) had strong DAT titers (1:˃102400), 2 (2.1%) demonstrated the lowest DAT titers (1:≤800), and 5 (5.4%) had marginal DAT titers (1:1600). Interestingly, the VL/HIV co-infected serum had a negative antibody titer (1:1600). Of the 6 VL/HBV co-infected sera, 1 (16.7%) and 5 (83.3%) demonstrated moderate (1:12800­1:51600) and strong (1:≥102400) DAT titers, respectively. The strong DAT titers observed in the VL/HBV co-infected sera were comparable to the DAT titers of the VL sera. The VL co-infection with HIV and hepatitis B/C is low in endemic areas in Eastern Sudan but may create a diagnostic difficulty. VL/HIV co-infected patients can have low Leishmania antibodies, thus alternative methodologies (e.g., antigen tests) may help the diagnosis.

Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs.

Control of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

Modifications in a Reference Freeze-Dried Direct Agglutination Test to Improve Visceral Leishmaniasis Detection.

Currently, a significantly lower temperature (35°C) than initially established (56°C) is indicated as the maximum temperature storage for the commercial reference visceral leishmaniasis (VL) freeze-dried direct agglutination test (FD-DAT). Despite an approximately 50% loss in the number of promastigotes in an FD-DAT batch that expired 7 years earlier, the promastigotes maintained a similar morphology to the equivalent valid batch implying most likely that auto-agglutination, rather than aging, is the main reason for expiry. The substitution of normal saline which was initially recommended for reconstitution, by citrate-saline/formaldehyde (CSF) as an anti-clumping/preservative agent resulted in restoration of validity comparable with that of the freeze-dried original or the liquid direct agglutination test (LQ-DAT) version (Friedman ANOVA test = 1.0588; P = 0.5890). Following a similar reconstitution procedure as for the 7-year expired antigen, using significantly lower promastigote concentration (1.4 × 107/mL) than in the non-expired (9.0 × 107/mL), good reliability for VL detection and stability at 4°C (> 12 months) were achieved. In comparison with the original version using normal saline ($32.0/vial), the cost-effectiveness of the FD-DAT was appreciably improved by the CSF incorporation and lowering of promastigote concentration per unit suspension medium ($12.8/vial). With diagnostic reliability comparable with the full-out titration used, FD-DAT procedure based on single sample dilution at the VL cutoff (1:3,200) permitted the use of significantly smaller antigen volumes (0.1 mL vs. > 1.5 mL), therefore contributing to a further reduction in the application cost. The successful replacement of ß-mercaptoethanol (ß-ME) by urea (T = 21.00; P = 0.0868) provided the required safety for the test procedure similar to the widely applied LQ-DAT.

Are We Now Well Prepared for Another Major Visceral Leishmaniasis Epidemic in Sudan?

To minimize the chance for future visceral leishmaniasis (VL) epidemics such as the 1988-1991 epidemic in Sudan, several VL detection tools have been introduced. There are many VL diagnostics with excellent sensitivities, specificities, and ease of use reported. However, additional test characteristics should be considered for use in the detection of future VL epidemics. The potential for local production or uninterrupted availability, low production and application costs, and stability at ≥45°C are of the utmost importance. Of the antibody-, antigen-, or DNA-based methods introduced, only a liquid direct agglutination test (LQ-DAT) remains in routine use. The LQ-DAT test may be the ideal diagnostic for detection of VL epidemics due to its low cost ($0.50/patient), stability under frequent and long-duration electric failures, and high level of reproducibility. The improved reliability for VL detection achieved locally through incorporating autochthonous L. donovani strains in antigen processing and precluding toxicants in test execution provides optimal sensitivity and safety for routine and mass application.

Identification of an area predominantly endemic for childhood and adolescent visceral leishmaniasis in central Sudan.

Although widely spread throughout Sudan, visceral leishmaniasis (VL) is predominantly endemic in the Gedaref, southern Blue-Nile, and Umrimta areas located in the eastern, southern, and central regions, respectively. Regardless of form (endemic or epidemic), VL occurrence follows similar patterns as all ages and both sexes are affected. From January 2005 to May 2016, we received a total of 563 patients with high suspicion for VL from various endemic areas; 159 were children and adolescents (0.5-18 years) from Umrimta (central Sudan). A significant observation during this 11-year period of uninterrupted monitoring using a standard liquid direct agglutination test (LQ-DAT) version was the exclusive VL occurrence (100%) in the child and adolescent populations of Umrimta when compared with other endemic areas (27.3%-48.0%). Among 12 child and adolescent suspects who initially tested marginal in the standard LQ-DAT, 6 scored unequivocally positive readings both in an improved LQ-DAT version (based on an autochthonous Leishmania donovani strain) and rK28 VL reference test. None of the 4 (2.5%) VL adult suspects (≥19years) referred had positive outcomes in the improved LQ-DAT version or the VL reference freeze-dried direct agglutination and rK28 tests. Further incorporation of antigens derived from autochthonous L. donovani strains from Umrimta (central Sudan) or Gedaref (eastern Sudan) in LQ-DAT significantly increased the agglutination titer levels in the respective VL homologous sera (p=0.0263 T=505 and p=0.2814T=219), suggesting possible antigenic variation within the predominant Sudanese L. donovani complex. Additional research is required to determine characteristics other than the serologically-based ones reported for the L. donovani strain involved.

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.

Beta-mercaptoethanol-modified ELISA for diagnosis of visceral leishmaniasis.

Following antigen preparation procedures similar to those of the direct agglutination test (DAT), an IgG ELISA employing intact beta-mercaptoethanol (beta-ME)-treated Leishmania donovani promastigotes was developed. The performance of the beta-ME ELISA thus developed was assessed in patients with confirmed visceral leishmaniasis (VL), revealing slightly lower sensitivity (39/40=97.5%) than that of the DAT (40/40=100%). When challenged with sera of individuals with non-VL conditions, including leukaemia and African trypanosomiasis, the specificity of the beta-ME ELISA was 100% (158/158), compared to 98.8% (156/158) for DAT. In an endemic population (n=145) manifesting a clinical suspicion of VL, results obtained with the beta-ME ELISA were highly concordant with those of DAT, both in the seropositive (65/68=95.6%) and seronegative (77/80=96.3%) groups. Furthermore, the incorporated intact antigen demonstrated higher sensitivity in ELISA (16/18=88.9%) than the water-soluble equivalent (13/18=72.2%). The stability of the formaldehyde-fixed antigen (2 months at 4 degrees C) in beta-ME ELISA, as well as the option for direct testing of whole-blood samples and visual reading of results (within 2 h, compared to 18 h for DAT), advocate the simultaneous application of the technique with DAT for confirmation of VL in laboratories with limited facilities.

Evaluation of a glycerol-preserved antigen in the direct agglutination test for diagnosis of visceral leishmaniasis at rural level in eastern Sudan.

Three-hundred and eight patients with suspected visceral leishmaniasis (VL) were received at Doka Hospital (eastern Sudan) during the period September 2004 to October 2005. The sensitivity and specificity of a glycerol-preserved (GP) antigen for VL diagnosis was assessed against the results of repeated lymph node aspiration and readings from a direct agglutination test (DAT) employing standard formaldehyde-fixed (FF) or freeze-dried (FD) antigen. Despite 13 months of storage at ambient temperature (28-47 degrees C), the GP antigen mean titres obtained from these 308 patients were no different from those that were FD (P=0.945) and stored under similar conditions, but were significantly different (P=0.019) from those that were FF and kept continuously at the optimum temperature for storage (4-8 degrees C). Taking the parasitological result as the gold standard and using a pre-established titre of 1 : 3200 as the DAT cut-off, the GP antigen revealed a sensitivity (91/105, 86.7 %) and specificity (187/203, 92.1 %) comparable to that of FD antigen (92/105, 87.6 %, and 188/203, 92.6 %, respectively) and FF antigen (94/105, 89.5 %, and 188/203, 92.6 %, respectively). At a titre range of 1 : 400-1 : 800, statistically determined as the optimum cut-off for the three antigens, sensitivities of 92.4, 90.5 and 96.2 % and specificities of 90.6, 90.1 and 88.7 % were achieved for the GP, FD and FF antigens, respectively, at a peripheral hospital. Regardless of the antigen preparation used, DAT results obtained in the peripheral hospital were highly reproducible in the central laboratory in Omdurman (weighted kappa: GP=0.957, FD=0.979 and FF=0.936). With a diagnostic reliability comparable to formaldehyde fixation and stability under ambient conditions similar to freeze drying, glycerol preservation, by virtue of its high potential for reproduction, meets the requirements for the management of VL in developing countries.