Immunological Testing Reveals Exposure to Malaria in the Hypoendemic Region of Iran.

Background. South eastern parts of Iran remain endemic for malaria infection. There is some concern that malaria infection may spread into Bushehr, which is located in the south western part bordering the Persian Gulf and at the periphery of the declared endemic region Hormozgan province due to frequency of visitors from eastern endemic areas and from neighboring malaria endemic countries. We investigated malaria prevalence in Bushehr. Methods and Results. Attempts were made to identify malaria active infection in blood smears and malaria specific antibody and antigens in serum samples. Traditional blood smears prepared from 1955 blood specimens yielded no definitive malaria positive case by microscopic technique. A total of 270 (13.8%) serum samples were positive for malaria antibodies. Using specific ELISA kits, presence of histidine rich proteins and lactate dehydrogenase antigens were investigated in serum samples. No histidine rich proteins specific for P. falciparum were detected amongst 270 antibody positive samples. However, six samples representing 0.3% of total population, were found to be positive for plasmodium pan specific lactate dehydrogenase antigens. This suggested the possibility of low level exposure to malaria in Bushehr community. Conclusions. Out of a total of 1955 samples tested, 270 (13.8%) were positive for malaria antibodies and six (0.3%) of these were positive for plasmodium-specific lactate dehydrogenase antigen suggesting a low level exposure to malaria in a hypoendemic region based on immunological testing. Since none of the 270 antibody samples were positive for histidine rich protein antigens, there is scope for further testing of blood samples by molecular methods such as polymerase chain reactions to confirm the plasmodium species and provide information valuable for future investigations. Our testing strategy for hypoemdemic malaria can be used as a template for investing malaria in 32 eliminating countries for testing ongoing transmission. This approach may be useful as a method in epidemiological studies.

Unified parasite lactate dehydrogenase and histidine-rich protein ELISA for quantification of Plasmodium falciparum.

There is a need for more objective and quantitative tools to replace microscopy in malaria diagnosis. Emphasis has recently been placed on alternative methods such as immunochromatography-based rapid tests. However, these tests provide only qualitative results. Two bio-molecules, parasite lactate dehydrogenase (pLDH) and histidine-rich proteins (HRPs), that are released by the intra-erythrocytic stages of the parasite offer certain specific characteristics that could potentially improve malaria diagnosis. In this paper, we describe a protocol for a unified sandwich ELISA that allows for the separate but concurrent measurement of pLDH and HRP biomolecules in aliquots taken from the same samples. Freshly drawn blood from a healthy unexposed adult male was used to serially dilute in vitro cultivated and synchronized ring stage Plasmodium falciparum parasites. Commercially available ELISA formats were modified to allow for the measurement of pLDH and HRP from aliquots of the same samples. The pLDH and HRP levels in the samples spiked with known numbers of infected red blood cells (iRBCs) were measured, and the values were used to generate standard graphs. The standard graphs were used to estimate the numbers of iRBCs in test samples. Serially diluted recombinant proteins were similarly used to generate a calibration curve, allowing for the expression of test results in nanograms of their respective recombinant protein. Levels of pLDH and HRPs were determined by using 1) P. falciparum culture material (cells and medium) 2) P. falciparum infected human blood (N = 6) samples, and 3) plasma from P. falciparum-infected patient (N = 22) samples. The parasite density of all culture and infected patient samples was also estimated by microscopy. Both pLDH and HRP levels correlated positively with the parasite density assessed by microscopy: Pearson correlation coefficient pLDH (r = 0.754, P < 0.0001, 95% CI: 0.47-0.89); HRP (r = 0.552, P < 0.007, 95% CI: 0.16-0.79). The HRPs seem to be released in larger quantities than pLDH (in a ratio of ~1 pLDH:~6 HRP), making the detection of HRP in culture material, blood, and plasma easier. The modified ELISA assay with quantitative measurement of pLDH and HRPs may provide a valuable tool for malaria research and patient management.

A novel exo-antigen-based ELISA for the detection of canine leishmaniasis.

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

The utility of exoantigens for detection of Leishmania infections.

Exoantigens released by Leishmania promastigotes were the subject of a workshop held in Mombasa, Kenya. Investigators from the Walter Reed Army Institute of Research (Silver Spring, Maryland) met with scientists from government and academic institutes and industry to review the current global status of leishmaniasis and to explore the potential role of exoantigens in the detection of Leishmania in the vertebrate host and arthropod vector. Some encouraging data, particularly in the immunodiagnosis of leishmaniasis, were shared. The participants concluded that the meeting provided a unique opportunity for investigators working on various aspects of the problem to network and to forge productive collaborations that could potentially lead to the development of more-effective tools to counter this persistent and expanding threat. They recommended periodic meetings to assess interval progress, to revise timelines, and to set achievable goals. The meeting also highlighted the importance of Leishmania infection in the 21st century, with more movement of people from disease-endemic to non-disease-endemic countries. Increased incidence and geographic spread of leishmaniasis emphasize the need for better and more reliable detection methods. Exoantigen-based diagnostic devices hold promise in this direction.

Assessment of assay sensitivity and precision in a malaria antibody ELISA.

Many types of ELISA-based immunodiagnostic test kits are commercially available in the market for specific indications. These kits provide necessary assay components, reagents, and guidelines to perform the assay under designated optimal conditions. By using these kits, any unknown or test sample can be assessed as negative or positive based on the results of referral calibrator (Ref+ve and Ref-ve) samples. It is essential to provide reliable test kits to end-users with adequate quality control analysis. Therefore, it is necessary to check the kit for any variations in its performance. While developing a malaria antibody ELISA test-kit, we optimized assay conditions with chequer-board analyses and developed an assay protocol. We have taken out kits randomly from the assembly line and had them evaluated by operators who are new to the test-kits. Assays are performed as per the test guidelines provided. Sera, diluted serially, have shown a clear discriminatory signal between a negative vs. positive sample. A COV is determined by evaluating the Ref-ve calibrator in replicate antigen-coated wells from 6 different plates. This COV is used as a tool to determine S/N ratio of test samples. Besides Ref-ve and Ref+ve calibrators, additional field serum samples are tested with the test kit. Several performance indices, such as mean, standard deviation, %CV are calculated, and the inter- and intra-assay variations determined. The assay precision is determined with large and small replicate samples. In addition, assays are performed concurrently in triplicate-, duplicate-, and single-wells, and the results are analyzed for any assay variations. Different plate areas are identified in antigen-coated 96-well plates and tested blind to detect any variations. The S/N ratio is found to be a very effective tool in determining the assay sensitivity. The %CV was within 10-15%. Variations seen in the assays are found to be due to operator errors and not due to kit reagents. These observations, although, are based upon one type; however, it may as well apply to other line of kits. This is obviously valuable to the end-users of ELISA kits. The operator related error has to be ascertained before lodging any complaint on the kit performance. Based on this data, the test kit has shown acceptable sensitivity and precision and offers compliance on the way the test kits is manufactured. With this, it is concluded that the test kits are suitable for detecting malaria antibody in clinical sample analysis.

Enzyme-linked immunosorbent assay based on soluble promastigote antigen detects immunoglobulin M (IgM) and IgG antibodies in sera from cases of visceral and cutaneous leishmaniasis.

Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity.