Accuracy of the direct agglutination test for diagnosis of visceral leishmaniasis: a systematic review and meta-analysis.

BACKGROUND: Parasitological investigation of bone marrow, splenic or lymph node aspirations is the gold standard for the diagnosis of visceral leishmaniasis (VL). However, this invasive test requires skilled clinical and laboratory staff and adequate facilities, and sensitivity varies depending on the tissue used. The direct agglutination test (DAT) is a serological test that does not need specialised staff, with just minimal training required. While previous meta-analysis has shown DAT to have high sensitivity and specificity when using parasitology as the reference test for diagnosis, meta-analysis of DAT compared to other diagnostic techniques, such as PCR and ELISA, that are increasingly used in clinical and research settings, has not been done. METHODS: We conducted a systematic review to determine the diagnostic performance of DAT compared to all available tests for the laboratory diagnosis of human VL. We searched electronic databases including Medline, Embase, Global Health, Scopus, WoS Science Citation Index, Wiley Cochrane Central Register of Controlled Trials, Africa-Wide Information, LILACS and WHO Global Index. Three independent reviewers screened reports and extracted data from eligible studies. A meta-analysis estimated the diagnostic sensitivity and specificity of DAT. RESULTS: Of 987 titles screened, 358 were selected for full data extraction and 78 were included in the analysis, reporting on 32,822 participants from 19 countries. Studies included were conducted between 1987-2020. Meta-analysis of studies using serum and DAT compared to any other test showed pooled sensitivity of 95% (95%CrI 90-98%) and pooled specificity of 95% (95%CrI 88-98%). Results were similar for freeze-dried DAT and liquid DAT when analysed separately. Sensitivity was lower for HIV-positive patients (90%, CrI 59-98%) and specificity was lower for symptomatic patients (70%, CrI 43-89%). When comparing different geographical regions, the lowest median sensitivity (89%, CrI 67-97%) was in Western Asia (five studies). CONCLUSIONS: This systematic review and meta-analysis demonstrates high estimated pooled sensitivity and specificity of DAT for diagnosis of VL, although sensitivity and specificity were lower for different patient groups and geographical locations. This review highlights the lack of standardisation of DAT methods and preparations, and the lack of data from some important geographical locations. Future well-reported studies could provide better evidence to inform test implementation for different patient populations and use cases. PROSPERO REGISTRATION: CRD42021240830.

Characterization of agglutinating antibodies detected by the direct agglutination test for visceral leishmaniasis diagnosis.

This study aimed to characterize agglutinating antibodies detected by the direct agglutination test (DAT-LPC) for the diagnosis of visceral leishmaniasis (VL). The DAT-LPC antigen/antibodies complex was recovered, washed, and used as antigenic substrate in a modified enzyme-linked immunosorbent assay (modified ELISA), revealed with anti-human IgM, IgG, and IgG subtype conjugates, and in the immunofluorescent antibodies test (IFAT), revealed with anti-human IgG and IgG1 conjugates. IgM antibodies were detected in 50%, IgG and IgG1 in 100%, and IgG3 in 52.8% of the 36 samples from VL patients. IFAT showed that agglutinating IgG and IgG1 antibodies recognized more intensely antigens located in the membrane and kinetoplast of the parasite. No antibodies were detected in the 15 samples from healthy individuals. This study shows for the first time that the antibodies responsible for agglutination in DAT-LPC are mostly of the IgG1 subtype.

Validation of in-house liquid direct agglutination test antigen: the potential diagnostic test in visceral Leishimaniasis endemic areas of Northwest Ethiopia.

BACKGROUND: Visceral leishmaniasis in Ethiopia is a re-emerging threat to public health, with increased geographical distribution and number of cases. It is a fatal disease without early diagnosis and treatment; thus, the availability of affordable diagnostic tools is crucial. However, due to delays caused by import regulations, procurement and late delivery of imported test kits, accessibility remains a problem in the control program. Therefore, we aimed to produce and evaluate the performance of an in-house liquid (AQ) direct agglutination test (DAT) antigen. RESULT: The AQ-DAT was produced at the Armauer Hansen Research Institute, using Leishmania donovani strain (MHOM/ET/67/L82). Sera from 272 participants; 110 microscopically confirmed cases of VL, 76 apparently healthy and 86 patients who had infectious disease other than VL were tested with AQ-DAT, and standard kits: Freeze-dried DAT (FD-DAT) and rK39. Taking microscopy as a gold standard; the sensitivity and specificity of the AQ-DAT were 97.3 and 98.8%, respectively. It had high degrees of agreement (k > 0.8), with a significant (P < 0.05) correlation compared to microscopy, FD-DAT, and rK39. CONCLUSION: Although further standardization is required, the in-house AQ-DAT could improve diagnostic accessibility, minimize intermittent stock outs and strengthen the national VL control program.

The diagnostic accuracy of direct agglutination test for serodiagnosis of human visceral leishmaniasis: a systematic review with meta-analysis.

BACKGROUND: Direct agglutination test (DAT) as a simple, accurate and reliable method, has been widely used for serodiagnosis of visceral leishmaniasis (VL) during the last three decades. The present study is a systematic review and meta-analysis to evaluate the diagnostic accuracy of DAT for serodiagnosis of human VL. METHODS: Electronic databases, including MEDLINE (via PubMed), SCOPUS, Web of Science, SID and Mag Iran (two Persian scientific search engines) were searched from December 2004 to April 2019. We determined the pooled sensitivity and specificity rates of DAT for the diagnosis of human VL, calculated positive and negative likelihood ratios (LR+ and LR-), and constructed summary receiver operating characteristic (ROC) curves parameters across the eligible studies. RESULTS: Of the 2928 records identified in the mentioned electronic databases and after examining reference lists of articles, 24 articles met inclusion criteria and were enrolled in the systematic review and out of them 20 records qualified for meta-analysis. The pooled sensitivity and specificity rates of DAT was 96% [95% CI, 92-98] and 95% [CI95% 86-99], respectively. The likelihood ratio of a positive test (LR+) was found to be 21 [CI95%, 6.6-66.5] and the likelihood ratio of a negative test (LR-) was found to be 0.04 [(CI95%, 0.02-0.08]. The combined estimate of the diagnostic odds ratio for DAT was high [467 (CI95%, 114-1912]). We found that the summary receiver operating characteristic curve (SROC) is positioned near the upper left corner of the curve and the area under curve (AUC) was 0.98 (95% CI, 0.97 to 0.99). CONCLUSION: Referring to our analysis, we determined that DAT can be considered as a valuable tool for the serodiagnosis of human VL with high sensitivity and specificity. As DAT is a simple, accurate and efficient serological test, it can be recommended for serodiagnosis of human VL particularly in endemic areas.

Seroprevalence and risk factors of Toxoplasma gondii infection in humans in East Hararghe Zone, Ethiopia.

A cross-sectional study was conducted from April 2013 to September 2013 to determine the seroprevalence and possible risk factors for human Toxplasma gondii infection in East Hararghe Zone, Ethiopia. Serum samples were analysed using direct agglutination test, and immunosorbent agglutination assay for detecting IgG (n = 354) and IgM (n = 167) T. gondii antibodies. The T. gondii IgG and IgM seroprevalences were 65·8% [95% confidence interval (CI) 60·62-70·75] and 8·98% (95% CI 5·11-14·38), respectively. Gender difference in IgG seroprevalence was not significant (P > 0·05), but 69·5% of adults exhibited an IgG seroresponse to T. gondii. Pregnant women showed 76·4% and 9·3% seropositivity to IgG and IgM antibodies, respectively. Multivariable logistic regression analysis identified the risk factors significantly associated with T. gondii seropositivity were district [odds ratio (OR) 2·24, 95% CI 1·25-4·01, P = 0·007], pipe water source (OR 6·70, 95% CI 2·70-16·64, P < 0·001), age, with adults (OR 4·32, 95% CI 1·91-9·75, P < 0·001), and keeping cats in the home (OR 2·01, 95% CI 1·11-3·65, P = 0·021). The high seroprevalence of toxoplasmosis in the human population in the study area and the corresponding level of IgM seropositivity may be indicative of reactivation or recent infection and further studies on the status of congenital toxoplasmosis in the study area merit consideration.

Seroprevalence of visceral leishmaniasis among pregnant women in Jahrom city in Fars province, southern Iran.

Background: Visceral leishmaniasis (VL) is a public health issue in endemic countries with poor sanitation facilities. In this study, the seroprevalence rate and associated risk factors of VL were investigated during September 2020 to February 2021 in pregnant women referred to Ostad Mottahari and Peymanieh hospitals in Jahrom county, Fars province, southern Iran. Material and methods: A total of 220 serum samples of pregnant women were assessed for the presence of Anti-Leishmania infantum antibodies by direct agglutination antigen (DAT). The associated risk factors were obtained using questionnaires. Results: The overall seroprevalence of VL in pregnant women was 12.72% (28/220). Considering the antibody titer, titer 1:1600 was detected in 23 samples, titer 1:3200 in 4 samples, and titer 1:6400 in one sample. All 5 women with titer >3200 had mild fever. As such, there was a statistically significant difference regarding the age (≥39 years old with p-value: 0.01). Conclusions: We recommend an appropriate health education program for pregnant women and serological screening of VL before pregnancy in endemic cities. Moreover, we believed a need for more epidemiological studies for better understand the status of VL in pregnant women.

Demographic characteristics and prevalence of asymptomatic Leishmania donovani infection in migrant workers working in an endemic area in Northwest Ethiopia.

Introduction: Visceral leishmaniasis (VL), a neglected tropical disease that causes substantial morbidity and mortality, is a serious health problem in Ethiopia. Infections are caused by Leishmania (L.) donovani parasites. Most individuals remain asymptomatic, but some develop VL, which is generally fatal if not treated. We identified the area of Metema-Humera in Northwest Ethiopia as a setting in which we could follow migrant workers when they arrived in an endemic area. The demographic characteristics of this population and factors associated with their risk of asymptomatic infection are poorly characterised. Methods: We divided our cohort into individuals who visited this area for the first time (first comers, FC) and those who had already been in this area (repeat comers, RC). We followed them from the beginning (Time 1, T1) to the end of the agricultural season (Time 2, T2), performing tests for sand fly bite exposure (anti-sand fly saliva antibody ELISA) and serology for Leishmania infection (rK39 rapid diagnostic test and the direct agglutination test) at each time point and collecting information on risk factors for infection. Results: Our results show that most migrant workers come from non-endemic areas, are male, young (median age of 20 years) and are farmers or students. At T1, >80% of them had been already exposed to sand fly bites, as shown by the presence of anti-saliva antibodies. However, due to seasonality of sand flies there was no difference in exposure between FC and RC, or between T1 and T2. The serology data showed that at T1, but not at T2, a significantly higher proportion of RC were asymptomatic. Furthermore, 28.6% of FC became asymptomatic between T1 and T2. Over the duration of this study, one FC and one RC developed VL. In multivariable logistic regression of asymptomatic infection at T1, only age and the number of visits to Metema/Humera were significantly associated with asymptomatic infection. Conclusion: A better understanding of the dynamics of parasite transmission and the risk factors associated with the development of asymptomatic infections and potentially VL will be essential for the development of new strategies to prevent leishmaniasis.

Comparison of the Diagnostic Performances of Five Different Tests in Diagnosing Visceral Leishmaniasis in an Endemic Region of Ethiopia.

The lack of accurate and feasible diagnostic tests poses a significant challenge to visceral leishmaniasis (VL) healthcare services in endemic areas. To date, various VL diagnostic tests have been or are being developed, and their diagnostic performances need to be assessed. In the present study, the diagnostic performances of rk39 RDT, the direct agglutination test (DAT), microscopy, loop-mediated isothermal amplification (LAMP), and miniature direct-on-blood polymerase chain reaction-nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) were assessed using quantitative polymerase chain reaction (qPCR) as the reference test in an endemic region of Ethiopia. In this study, 235 suspected VL cases and 104 non-endemic healthy controls (NEHCs) were recruited. Among the suspected VL cases, 144 (61.28%) tested positive with qPCR. The sensitivities for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA were 88.11%, 96.50%, 76.58%, 94.33%, and 95.80%, respectively. The specificities were 83.33%, 97.96%, 100%, 97.38%, and 98.92% for rk39 RDT, DAT, microscopy, LAMP assay, and mini-dbPCR-NALFIA, respectively. In conclusion, rk39 RDT and microscopy exhibited lower sensitivities, while DAT demonstrated excellent performance. LAMP and mini-dbPCR-NALFIA showed excellent performances with feasibility for implementation in remote endemic areas, although the latter requires further evaluation in such regions.

Visceral Leishmaniasis in Iran: An Update on Epidemiological Features from 2013 to 2022.

Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. The zoonotic form of VL is endemic in some areas of Iran. We aimed to determine the status of VL identified in humans and canines in different parts of Iran from 2013 to 2022. Method: A national representative cross-sectional study was conducted in 10 provinces of Iran, including the national leishmaniasis reference lab. We employed the direct agglutination test (DAT) as a reliable serological method to detect anti-Leishmania infantum antibodies in humans and animal reservoir hosts. Additionally, a narrative literature review was conducted to identify relevant studies on VL seroprevalence in Iran from 2013 to 2023. Results: The results of 21281 human and 5610 canine serum samples from 2013 to 2022 are reported. Altogether, 448 (2.1%, 95%CI: 2.0-2.3) human serum samples showed anti-L. infantum antibody levels of ≥1:3200. Of these samples, 13716 (64.5%) were collected actively, which showed a seroprevalence of 0.6% (95% CI: 0.5-0.8) and 7565 (35.5%) were collected passively, which showed a seroprevalence of 4.8% (95%CI: 4.3-5.3). Overall, 1035 (20.1%, 95%CI: 19.0-21.2) of 5160 domestic dogs (Canis familiaris) samples showed anti-L. infantum antibody levels of ≥1:320. Northwest (2.8%) and northeast (0.96%) regions had the highest human VL seroprevalence, while northwest (21.5%) and south (14.4%) regions had the highest canine VL seroprevalence. Conclusion: Zoonotic VL, an endemic parasitic disease, is still present in several different distinct areas across Iran. While human VL cases have shown a declining trend over the last decade, the prevalence of canine VL remains significant.

Iron Deficiency Anemia Presenting With Pancytopenia: A Study From India.

INTRODUCTION: Iron deficiency anemia (IDA) is the most common cause of anemia worldwide. IDA is commonly associated with thrombocytosis and normal or slightly decreased leukocyte count. Sometimes it can present with thrombocytopenia, but rarely present with pancytopenia. Here we are presenting six cases of severe iron deficiency presenting with pancytopenia, which responded to iron replenishment. METHODS:  This 12-month observational study was conducted in the Department of General Medicine at a tertiary care Centre in India. All cases of pancytopenia (after exclusion of other causes) with IDA were included. IDA was established with the help of a complete blood count (CBC), peripheral smear examination, serum iron studies, and serum ferritin.  Results: In our study, CBC at four weeks later of iron transfusion without other supplementation showed significant improvement in hematological parameters. CONCLUSION:  Severe iron deficiency is a reversible etiology of pancytopenia. It should be kept as a differential diagnosis of pancytopenia if common causes of pancytopenia are ruled out.

Asymptomatic Leishmania donovani infection and associated factors among blood donors attending at Metema district Blood Bank, Northwest Ethiopia: a cross- sectional study.

BACKGROUND: Ethiopia is one of the top 10 countries in the world where 90% visceral leishmaniasis cases are reported. Metema-Humera lowlands are the most important foci in Ethiopia. Blood transfusion in visceral leishmaniasis endemic foci in Ethiopia does not consider screening of visceral leishmaniasis during blood donation. The aim of this study is therefore, was to assess asymptomatic Leishmania donovani infection and associated factors among blood donors attending at Metema district Blood Bank, Northwest Ethiopia. METHODS: A Health facility based cross-sectional study was conducted at Metema Blood Bank from February to March 2020. A total of 205 blood donors were eligible and participated in this study. Structured questionnaire were used to collect data on socio-demographic characteristics and perceived risk factors associated with asymptomatic visceral leishmaniasis among blood donors. Blood donors were screened using both rK39 and direct agglutination tests based on the manufactures' instructions. Data were analyzed using SPSS version 20.0. Chi-square test was used to assess associations of Leishmania donovani infection with predisposing factors. Associations were considered statstically significant on P-value < 0.05 at 95% confidence level. RESULTS: Of the total 205 participants, 32(15.6%) were positive for asymptomatic Leishmania donovani infection at least by one of the diagnostic tests used. Eight (3.9%) and 30(14.6%) of the participants` were positive by the rK39 and direct agglutination tests, respectively. Six (2.9%) donors were tested positive by both diagnostic tests. Family history of visceral leishmaniasis (X²=11.334, P = 0.003) and having neighbors with history of visceral leishmaniasis (X²=5.923, P = 0.015) were significantly associated with asymptomatic Leishmania donovani infection among blood donors. CONCLUSIONS: The prevalence of asymptomatic Leishmania donovani infection was 15.6%. Asymptomatic visceral leishmaniasis was significantly associated with donors' family and neighbors' history of infection. Therefore, laboratory screening of blood donors for visceral leishmaniasis in endemic areas will be mandatory. Moreover, this study will give base line information for future study in the country.

A Sero-Epidemiological Study on Visceral Leishmaniasis among Volunteer Children and Adults in Rural Areas of Shahroud, Iran 2018-2019.

Background: Visceral leishmaniasis (VL) also known as Kala-azar is considered as one of the zoonotic infections in Mediterranean countries. The disease reservoir and vectors are dogs and sandflies respectively. Due to reported sporadic cases of Kala-azar in the past five years in Shahroud County, Semnan Province, Iran, this study aimed to investigate the status of this infection in this area and to determine its seroepidemiology to take required measurements for infection control and treatment. Methods: This study was conducted on 504 subjects residing in seven villages in Shahroud County. Blood samples were randomly selected using the cluster sampling method and were collected from subjects aged up to 13 years old (90%) and adults over 13 years old (10%) from September to May 2019. After separating sera from whole blood, samples were subjected to direct agglutination test (DAT) to detect anti-Leishmania infantum antibodies. A range of 1:10 to 1:800 dilutions were prepared from the samples. Results: Results of 1:800 titration indicated that no sample was positive for antibodies against L. infantum. After the secondary screening, 10 cases (1.98%) showed the antibody titer of 1:100, while four cases (0.79%) showed the antibody titer of 1:400. Of 14 cases with the L. infantum antibodies, all were detected from the children <13 years old. According to clinical findings, no patient was suffering from fever, weight loss, splenomegaly, hepatomegaly, and cachexia and therefore did not show the Kala-azar symptoms. Conclusion: The results of the current study indicate that Kala-azar is not prevalent in Shahroud County.

Seroepidemiological Study of Visceral Leishmaniasis (Kala-Azar) in Children under 12 Years Old in North of Iran: An Observational Study in 2019-2020.

Background: Visceral leishmaniasis (VL) caused by the Leishmania donovani complex that is transmitted by the bites of female sandflies. Mediterranean type of VL caused by L. infantum. While, Roudbar County of Guilan Province has been introduced as a suspected cutaneous leishmaniasis focus; there are no published data on the seroprevalence of VL in Guilan Province. We aimed to investigate the sero-prevalence of this disease in Roudbar County. Methods: This descriptive cross-sectional study was carried out in 2019-2020 among children less than 12 years of age to determine the seroprevalence of VL by direct agglutination test (DAT). Blood samples were randomly collected from 918 children under 12 years of age refers to the public health center in the clusters. Results: Out of 918 children, 14 (1.52%) showed anti-Leishmania antibodies, with 4 (0.43%), 2 (0.21%), 8 (0.87%) anti-L. infantum antibodies at titers 1:800, 1:1600 and ≥1: 3200 respectively. All children with anti-Leishmania antibody titers of ≥1:800 were evaluated by a physician. Clinical manifestation of VL including fever, anemia and hepatosplenomegaly observed in a 6-year-old boy from Defraz village with anti-Leishmania antibody of titers ≥102400.This patient was admitted to the pediatric hospital in Rasht, capital of Guilan province, Iran and was successfully treated. Conclusion: VL is being circulated with low prevalence in children up to 12 years old in Roudbar, northern part of Iran. Accordingly, it is critical to improve the awareness of physicians and public health supervisors about the importance of this fatal disease in Guilan province and especially in Roudbar area.

Seroprevalence and Risk Factors Associated with Leishmania Infection in Dogs from Portugal.

Canine leishmaniosis (CanL) caused by Leishmania infantum is an important zoonosis in southwestern European countries where this disease is endemic, and dogs, as domestic animals in close contact with humans, are the reservoir hosts for the parasite. In Portugal, CanL is of relevant veterinary concern. The previous national study revealed an overall seroprevalence of 6.3%. Since then, new prophylactic measures, such as vaccines, have been introduced in Europe. The aim of this study was to update seroprevalence for Leishmania infection and reassess risk factors in Portugal. A cross-sectional study was conducted from January-March 2021 with 1860 client-owned dogs from continental Portugal. A questionnaire and whole blood samples on filter paper were collected and a direct agglutination test was used to calculate anti-Leishmania antibody titres. True seroprevalence was 12.5% (95% CI 10.3-13.2%). Potential risk factors associated with L. infantum infection in dogs were age ≥ 2 years (aOR = 1.68, 95% CI 1.1-2.6) and residing in the interior regions of the country (aOR = 1.92, 95% CI 1.3-2.9) and non-use of repellents (aOR = 1.75, 95% CI 1.2-2.5). The key to controlling CanL and its impact on Public Health in endemic areas lies in continuous implementation of prophylactic measures, through the correct use of repellents/insecticides and vaccines and early detection and monitoring of infected dogs.

Prevalence of asymptomatic visceral leishmaniasis in human and dog, Benishangul Gumuz regional state, Western Ethiopia.

BACKGROUND: The Benishangul-Gumuz region is an important development corridor in Ethiopia. Large-scale projects such as the Great Renaissance Dam, mining and agriculture have entailed huge environmental modifications and settlement pattern changes. There is no detailed epidemiological information on visceral leishmaniasis (VL) in the region. MATERIALS AND METHODS: A cross-sectional study was carried out to assess the epidemiology and risk factors associated with Leishmania infection. A leishmanin skin test (LST) was done for 1342 participants, and for 253 of them rK39 and DAT were carried out. Thirty-six dogs owned by households with LST-positive member(s) were rK39 and DAT tested. A pretested questionnaire was used to capture individual and household characteristics. RESULTS: Of the 89.2% (1197/1342) who availed themselves of the LST reading, 6.0% were positive. The rk39 and DAT positivity among the 253 tested were 3.2% and 5.9%, respectively. In dogs, positivity rates by rK39 and DAT were 13.9% and 5.6%, respectively. Of the household and individual risk factors, presence of a dog in the household (P = 0.005), male sex (0.003), residence woreda (0.000) and occupation (0.023) showed a strong positive association with LST positivity. Individuals who lived in households that had dogs were 2.6 times more likely to be LST positive (AOR = 2.6; 95% CI = 1.54, 4.40). Being female decreased the probability of being LST positive by 0.38 times (AOR = 0.38; 95% CI = 0.20, 0.72). People living in Guba and Kurmuk had 4.7 (AOR = 4.74, 95% CI 1.83, 12.31) and 5.9 (AOR = 5.85, 95% CI 2.27, 15.09) times more risk of being infected. CONCLUSIONS: We demonstrated the presence of active VL transmission in the areas. Thus, we underline the need to establish the responsible vector(s) and reservoir(s) for comprehensive early containment plans to prevent potentially harmful public health and economic consequences.

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis in Owned Dogs (Canis familiaris) in New Foci of Rural Areas of Alborz Province, Central Part of Iran: A Cross-Sectional Study in 2017.

BACKGROUND: Mediterranean form of visceral leishmaniasis (VL) is endemic among some provinces of Iran. The present study was designed to determine the prevalence of canine visceral leishmaniasis (CVL) in the owned dogs of the rural areas of Alborz Province near Tehran as the capital of Iran. METHODS: This study conducted on 303 owned dogs that selected using a stratified random sampling method. The direct agglutination test (DAT) was used to determine the frequency of Vl. The spleen biopsy was taken from the serology-positive dogs for the confirmation of CVL in the suspected dogs. Nested PCR and sequencing methods were used to determine the type of Leishmania species in the dogs which were parasitological positive. RESULTS: Overall, the DAT results of 9 dogs (2.97%, CI: 1.57-5.55) showed anti Leishmania antibodies at titers ≥ 1:320 indicating VL infection. One dog (0.33%, CI 95%: 0.06-1.85) showed clinical signs and symptoms of VL. There was a significant correlation between the positive cases of CVL and rural area (p< 0.001). The Leishmania was observed in the impression smears that were prepared from spleen biopsy of five the studied dogs. Leishmania infantum were confirmed in all them using nested-PCR assay. The sequence analysis of all five isolates was 95% similar to L. infantum. CONCLUSION: This study shows that domestic cycle of L. infantum has been established in rural areas of Alborz province where located near Tehran as capital city of Iran. It is necessary to increase the awareness and monitoring of the disease periodically.

User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and ß-mercaptoethanol in test execution.

PURPOSE: Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. METHODOLOGY: In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after ß-mercaptoethanol (ß-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. RESULTS: Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05 % (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with ß-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with ß-ME, was achieved by incorporating urea (0.3 % wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and ß-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL - Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. CONCLUSIONS: By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.

Canine Visceral Leishmaniasis; A Seroepidemiological Survey in Jiroft District, Southern Kerman Province, Southeastern Iran in 2015.

BACKGROUND: Domestic dogs have been implicated as the main reservoir host of Mediterranean type of visceral leishmaniasis (VL) that is endemic in some parts of Iran. This study was performed about role of dogs in canine VL (CVL) epidemiology in Jiroft District, south of Kerman Province, southeastern Iran. METHODS: Totally, 165 dogs including 100 stray and 65 sheepdogs were randomly selected. After complete clinical examination blood sample was taken from each dog. All the collected samples were examined following the serum separation by direct agglutination test (DAT) for detection of anti-Leishmania infantum antibodies. The titers of ≥1:320 were defined as positive. RESULTS: Overall, of 165 serum samples, 13 samples (7.9%) were positive by DAT at titers of ≥1:320. The seroprevalence was 11% among the stray dogs and 3% among the sheepdogs. There was no significant difference between stray and sheepdogs in CVL infection. The highest seroprevalence rate (14.3%) was found in seven-year old dogs. CONCLUSION: The present finding indicates the role of stray and sheepdogs in CVL epidemiology in this area. Further investigations are needed to evaluate the status of VL infection in human subjects in this area.

Identification of Clinical Immunological Determinants in Asymptomatic VL and Post Kala-azar Dermal Leishmaniasis Patients.

BACKGROUND: Visceral Leishmaniasis (VL) caused by protozoa belonging to the genus Leishmania, usually have anthroponotic mode of transmission and is issue of great public health importance in Indian subcontinent. Asymptomatic cases of VL and PKDL are subject of keen interest to find their role in the transmission of VL in epidemic areas. We evaluated the immunological cytokine determinants expressed in most clinical suspects of asymptomatic VL and PKDL (IL-10, IFN-γ, and TNF-α). METHODS: Eighty-four participants were included at RMRIMS, Patna, India in 2016-17 out of which 64 asymptomatic individual positive for rK-39, without sign and symptoms of VL; 15 PKDL patient's with past history of VL and 5 endemic healthy subjects were recruited from VL endemic areas. DAT and quantitative assessment of plasma cytokines was determined from the blood samples collected in a plain and sodium-EDTA vacutainer respectively from the subjects. RESULTS: The mean level of IL-10 in DATpos LOW of asymptomatic VL and PKDL was significantly higher than endemic healthy (P<0.05). The cytokine polarization index (IFN-γ versus IL-10) was significantly low in PKDL cases compared with asymptomatic VL cases in DATpos LOW titre (P<0.05). This index was low again but statistically not significant in PKDL than in asymptomatic VL when TNF-α was considered against IL-10. The ratio of IFN-γ: IL-10 and TNF-α: IL-10 was observed decreased both in asymptomatic VL and PKDL cases than in healthy from endemic areas. CONCLUSION: Collectively we surmise from our data that asymptomatic VL can also play an important role like PKDL in transmission of VL.

Assessment of Recombinant A2-Latex Agglutination Test (RA2-LAT) and RA2-ELISA for Detection of Canine Visceral Leishmaniasis: A Comparative Field Study with Direct Agglutination Test in Northwestern Iran.

BACKGROUND: This study aimed to set-up latex agglutination test (LAT) and ELISA based on recombinant A2 from Iranian strain of Leishmania (L.) infantum (rA2-Ag) and evaluated for detection of anti-Leishmania antibodies in dogs compared to standard direct agglutination test (DAT). METHODS: The rA2-Ag was synthesized under a part of the A2 gene sequences which contain immune dominant sequences and less number of repetitive sequences. Latex beads, 0.8 µm (Sigma, USA) were sensitized with rA2-Ag. The tests were carried out on sera collected from 350 ownership dogs including symptomatic (n=67), asymptomatic (n=230) canine visceral leishmaniasis (CVL), and (n=53) uninfected domestic dogs as control group. RESULTS: Anti-leishmanial antibodies were detected in 97 (27.7%), 96 (27.4%) and 29 (%9) of the serum samples by using DAT, rA2-ELISA, and rA2-latex, respectively with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A combined sensitivity of 52% and specificity of 82.40% for rA2-ELISA and 23.8% and specificity 95.38%, respectively were found with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. The concordance between rA2-ELISA and rA2 latex compared with DAT as a gold standard serological test for VL were found 73.7% and 77.5%, respectively. CONCLUSION: A good degree of agreement was found between rA2-ELISA and DAT (73.7%). rA2-ELISA could detect more seropositive serum samples than rA2-LAT and it may be recommended as an alternative tool for the diagnosis of CVL.