Diagnosis of cutaneous leishmaniasis in Guatemala using a real-time polymerase chain reaction assay and the Smartcycler.

The polymerase chain reaction (PCR) assay has been reported for the diagnosis of cutaneous leishmaniasis (CL). Real-time (RT) PCR offers several advantages over traditional PCR, including faster processing time and decreased risk of contamination. Enhanced portability is another benefit that expands the applicability of the assay. A portable RT-PCR assay was transported from the United States to Guatemala for comparison with traditional diagnostic modalities. With the clinical diagnosis of CL as the gold standard, RT-PCR was positive in 86% (37 of 43) versus 53% (20 of 38) for microscopy and 72% (28 of 39) for culture. Negative RT-PCR samples (6) were also negative by traditional diagnostic methods (although subsequently determined to be positive by a nested kDNA PCR). Sixty-four percent (9 of 14) of cases tested and negative by microscopy and/or culture were positive by RT-PCR. This study demonstrates that a RT-PCR assay can be successfully deployed to offer enhanced sensitivity for the diagnosis of CL.

Evidence for transmission of Plasmodium vivax among a duffy antigen negative population in Western Kenya.

We present evidence that a parasite with characteristics of Plasmodium vivax is being transmitted among Duffy blood group-negative inhabitants of Kenya. Thirty-two of 4,901 Anopheles gambiae and An. funestus (0.65%) collected in Nyanza Province were ELISA positive for the P. vivax circumsporozoite protein VK 247. All positives were found late in the rainy season, when An. funestus predominated, and disproportionately many were found at a single village. A P. vivax specific sequence of the SSU rRNA gene was amplified from three of six ELISA-positive mosquitoes. Erythrocytes from 31 children, including 9 microscopically diagnosed as infected with P. vivax, were negative by flow cytometry for the Fy3 or Fy6 epitopes, which indicate Duffy blood group expression. A DNA fragment specific for the C terminus of the gene for P. vivax merozoite surface protein 1 (MSP-1) was amplified from the blood of four of these children and subsequently sequenced from two.

Epidemiology and spatial analysis of malaria in the Northern Peruvian Amazon.

A retrospective surveillance study was conducted to examine the micro-geographic variation of malaria incidence in three malaria-endemic communities in the Northern Peruvian Amazon. The annual malaria risk rate (per 100) ranged from 38% to 47% for Plasmodium vivax and from 15% to 18% for P. falciparum. Spatial clusters were found for P. vivax in Padre Cocha, Manacamiri, and Zungaro Cocha, and for P. falciparum only in Padre Cocha. Spatial-temporal clusters showed that the highest monthly number of P. vivax cases varied every year from December to March in 1996-1997 and from February to June in 1998-1999, and for P. falciparum from November to April in 1996-1997 and from January to April in 1998-1999. Our results suggest a constant presence of high-risk areas (hot spots) for malaria infection in periods with high or low malaria incidence. Modest targeted control efforts directed at identified high-risk areas may have significant impact on malaria transmission in this region.

Spatial clustering and epidemiological aspects of visceral leishmaniasis in two endemic villages, Baringo District, Kenya.

Visceral leishmaniasis (VL) seroprevalence in Kenya is unknown because of the lack of a practical and accurate diagnostic test or surveillance system. A novel serological assay was used to estimate the seroprevalence of Leishmania-specific antibodies, and Global Information System and spatial clustering techniques were applied to study the presence of spatial clusters in Parkarin and Loboi villages in Baringo District in 2001. VL seroprevalences were 52.5% in Parkarin and 16.9% in Loboi. Significant associations among seropositivity and house construction, age, and proximity to domestic animal enclosures were found. A significant spatial cluster of VL was found in Loboi. The spatial distribution of cases in the two villages was different with respect to risk factors, such as presence of domestic animals. This study suggests that disease control efforts could be focused on elimination of sand fly habitat, placement of domestic animal enclosures, and targeted use of insecticides.

Evaluation of serological assays based on a novel excreted antigen preparation for the diagnosis of Cutaneous Leishmaniasis in Panama.

The objective of the present study was to determine the efficacy of prototype diagnostic serological assays for American Cutaneous Leishmaniasis (ACL) in Panama. As such, we prospectively sampled 100 cutaneous leishmaniasis case-patients and tested their sera in two serological assays based upon novel soluble antigen preparations made from propagating the parasites in a protein-free, serum free media. Using serum and a Leishmania mexicana antigen preparation to sensitize plates, the assay correctly identified 89% of the case-patients. While using serum with an antigen preparation from Leishmania braziliensis, the assay correctly identified 71% of the patients. Concerning both test formats, performance was near equal in true positive and presumptive positive subsets demonstrating the improved sensitivity of these assays over reference methods of choice. Since the incidence of leishmaniasis in Panama has increased dramatically in the past 10 years, these assays may be useful in clinical and epidemiological studies and control programs.

Evaluation of the VecTest Malaria Antigen Panel assay for the detection of Plasmodium falciparum and P. vivax circumsporozoite protein in anopheline mosquitoes in Thailand.

We evaluated the performance of the VecTest Malaria Antigen Panel (V-MAP) assay for the detection of Plasmodium falciparum and P. vivax (variants 210 and 247) circumsporozoite protein in anopheline mosquitoes in Thailand. The V-MAP assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. The circumsporozoite (CS) ELISA was used as the reference standard. Mosquitoes evaluated in the study included field-collected specimens (n = 930) and laboratory-reared specimens that had been fed on blood collected from patients with and without Plasmodium gametocytes (n = 4,110) or on cultured P. falciparum gametocytes (n = 262). Field-collected mosquitoes were triturated individually or in pools of 2-5 and tested using 613 V-MAP assays. Laboratory-reared specimens were tested individually using 4,372 V-MAP assays. Assay performance depended on the species of Plasmodium and the number of sporozoites used as the cut-off. For P. falciparum, optimal performance was achieved using a cut-off of 150 sporozoites (sensitivity = 100%, specificity = 99.2%, and accuracy = 0.99). For P. vivax variant 210, optimal performance was also achieved using a cut-off of 150 sporozoites (sensitivity = 94.8%, specificity = 94.5%, and accuracy = 0.95). We were unable to develop a standard-curve for the CS-ELISA using P. vivax variant 247 because of a lack of sporozoites; however, using a cut-off of 30 pg P. vivax 247 antigen (mosquitoes with less than this amount of antigen were considered negative), assay performance (sensitivity = 94.3%, specificity = 99.2%, and accuracy = 0.99) was comparable to that achieved for P. falciparum and P. vivax 210. These results clearly demonstrate that the V-MAP assay performs at an acceptable level and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

The domestic dog, a potential reservoir for Leishmania in the Peten region of Guatemala.

In the present study, domestic dogs in a Leishmania endemic area in the Peten Region of Guatemala were sampled to determine if they are a potential reservoir for Leishmania parasites. Blood from 100 dogs from six villages was tested with two different antibody-capture assays for Leishmania-specific antibodies and a 28% seroprevalence was determined. Tissue scrapings from six dogs presenting with chronic lesions characteristic of Leishmania infection were sampled and four dogs were positive by a genus-specific fluorogenic PCR assay. Histopathology by giemsa stain confirmed the presence of amastigotes in one of these dogs. These findings support the hypothesis that dogs may play an important role in the transmission of Leishmania in a region where no mammal has ever been implicated as a reservoir.

Sensitivity of the VecTest antigen assay for eastern equine encephalitis and western equine encephalitis viruses.

VecTest assays for detecting eastern equine encephalitis virus (EEE) and western equine encephalitis virus (WEE) antigen in mosquito pools were evaluated to determine their sensitivity and specificity by using a range of EEE, WEE, St. Louis encephalitis virus (SLE), and West Nile virus (WN) dilutions as well as individual and pooled mosquitoes containing EEE or WEE. The EEE test produced reliable positive results with samples containing > or = 5.3 log10 plaque-forming units (PFU) of EEE/ml, and the WEE test produced reliable positive results with samples containing > or = 4.7 log10 PFU WEE/ml. Both assays detected the respective viral antigens in single virus-positive mosquitoes and in pools containing a single positive mosquito and 49 negative specimens. The SLE and WN assays also contained on the dipsticks accurately detected their respective viruses. No evidence was found of cross reaction or false positives in any of the tests. The VecTest assays were less sensitive than the EEE- and WEE-specific TaqMan reverse transcriptase polymerase chain reaction and Vero cell plaque assay, but appear to be useful for detecting arboviruses in mosquito-based arbovirus surveillance programs.

Comparison of vero cell plaque assay, TaqMan reverse transcriptase polymerase chain reaction RNA assay, and VecTest antigen assay for detection of West Nile virus in field-collected mosquitoes.

Mosquitoes collected during the epidemic of West Nile virus (WN) in Staten Island, NY, during 2000 were identified to species, grouped into pools of up to 50 individuals, and tested for the presence of WN by using TaqMan reverse transcriptase polymerase chain reaction (RT-PCR) to detect West Nile viral RNA, Vero cell plaque assay to detect infectious virus, and VecTest WNV/SLE Antigen Panel Assay. A total of 10,866 specimens was tested in 801 pools. Analysis of results indicated that TaqMan RT-PCR detected 34 WN-positive pools, more than either of the other techniques. The plaque assay detected 74% of the pools positive by TaqMan, and VecTest detected 60% of the pools positive by TaqMan. The VecTest assay detected evidence of West Nile viral antigen in 67% of the pools that contained live virus detected by plaque assay. A WN enzyme immunoassay performed similarly to the VecTest WN assay. Differences in performance were related to relative sensitivity of the tests. Infection rates of WN in Culex pipiens and Cx. salinarius calculated by the 3 techniques varied, but each estimate indicated a high infection rate in the population. Positive and negative attributes of each procedure, which may influence how and where they are used in surveillance programs, are discussed.

Visceral leishmaniasis in a soldier returning from Operation Enduring Freedom.

This report presents a case of visceral leishmaniasis in a soldier returning from Operation Enduring Freedom. During the United States' last major military conflict, Operation Desert Storm, the diagnosis of multiple cases of visceral leishmaniasis led to policy changes, including a temporary ban on troop blood donation. This case demonstrates the applicability of recently developed Leishmania polymerase chain reaction and serological assays when conventional methods of diagnosis, such as tissue microscopy and culture, fail.

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.

Immunization with Leishmania major exogenous antigens protects susceptible BALB/c mice against challenge infection with L. major.

The potential of Leishmania major culture-derived soluble exogenous antigens (SEAgs) to induce a protective response in susceptible BALB/c mice challenged with L. major promastigotes was investigated. Groups of BALB/c mice were immunized with L. major SEAgs alone, L. major SEAgs coadministered with either alum (aluminum hydroxide gel) or recombinant murine interleukin-12 (rmIL-12), L. major SEAgs coadministered with both alum and rmIL-12, and L. major SEAgs coadministered with Montanide ISA 720. Importantly and surprisingly, the greatest and most consistent protection against challenge with L. major was seen in mice immunized with L. major SEAgs alone, in the absence of any adjuvant. Mice immunized with L. major SEAgs had significantly smaller lesions that at times contained more than 100-fold fewer parasites. When lymphoid cells from L. major SEAg-immunized mice were stimulated with leishmanial antigen in vitro, they proliferated and secreted a mixed profile of type 1 and type 2 cytokines. Finally, analyses with Western blot analyses and antibodies against three surface-expressed and secreted molecules of L. major (lipophosphoglycan, gp46/M2/PSA-2, and gp63) revealed that two of these molecules are present in L. major SEAgs, lipophosphoglycan and the molecules that associate with it and gp46/M2/PSA-2.