Rapid diagnosis of Leishmania infection with a portable loopmediated isothermal amplification device.

L. donovani is an intracellular protozoan parasite, that causes visceral leishmaniasis (VL), and consequently, post-kala azar dermal leishmaniasis (PKDL). Diagnosis and treatment of leishmaniasis is crucial for decreasing its transmission. Various diagnostic techniques like microscopy, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods are used to detect leishmaniasis infection. More recently, loop-mediated isothermal amplification (LAMP) assay has emerged as an ideal diagnostic measure for leishmaniasis, primarily due to its accuracy, speed and simplicity. However, point-of-care diagnosis is still not been tested with the LAMP assay. We have developed a portable LAMP device for the monitoring of Leishmania infection. The LAMP assay performed using our device can detect and amplify as little as 100 femtograms of L. donovani DNA. In a preliminary study, we have shown that the device can also amplify L. donovani DNA present in VL and PKDL patient samples with high sensitivity (100%), specificity (98%) and accuracy (99%), and can be used both for diagnostic and prognostic analysis. To our knowledge, this is the first report to describe the development and application of a portable LAMP device which has the potential to evolve as a point-of-care diagnostic and prognostic tool for Leishmania infections in future.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani.

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detect Leishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmania genus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence.

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 µm long and 6.6-15.7 µm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus.

Detection of Blastocystis from stool samples using real-time PCR.

We developed a real-time LC PCR assay to detect a 152 bp sequence in an uncharacterized region of the Blastocystis genome. The described assay detected 11 of 11 ATCC strains of Blastocystis from subtypes 1, 3, and 4. Three of three stool samples from Oregon and California military personnel that were negative for Blastocystis by an ova and parasite test as well as a conventional PCR assay were positive for Blastocystis using our real-time LC PCR assay. Diagnosis of Blastocystis infections using this sensitive method, including DNA extraction and real-time PCR, only requires 3 h. The lower limit of detection for Blastocystis in stool using the real-time LC PCR assay was calculated to be 760 cells of Blastocystis per 100 mg of stool, an estimated 760 parasites per reaction. The assay did not cross-react with Ruminococcus hansenii, Anarococcus hydrogenalis, Bifidobacterium adolescentis, Fusobacterium prausnitzii, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, or Lactobacillus acidophilus. Because of the ease of use, sensitivity, specificity, and increase in Blastocystis infections in the USA we believe this assay has the potential to be useful as a clinical diagnosis tool of Blastocystis infection.

Morphological variation and phylogenetic analysis of the dinoflagellate Gymnodinium aureolum from a tributary of Chesapeake Bay.

Cultures of four strains of the dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen were established from the Elizabeth River, a tidal tributary of the Chesapeake Bay, USA. Light microscopy, scanning electron microscopy, nuclear-encoded large sub-unit rDNA sequencing, and culturing observations were conducted to further characterize this species. Observations of morphology included: a multiple structured apical groove; a peduncle located between the emerging points of the two flagella; pentagonal and hexagonal vesicles on the amphiesma; production and germination of resting cysts; variation in the location of the nucleus within the center of the cell; a longitudinal ventral concavity; and considerable variation in cell width/length and overall cell size. A fish bioassay using juvenile sheepshead minnows detected no ichthyotoxicity from any of the strains over a 48-h period. Molecular analysis confirmed the dinoflagellate was conspecific with G. aureolum strains from around the world, and formed a cluster along with several other Gymnodinium species. Morphological evidence suggests that further research is necessary to examine the relationship between G. aureolum and a possibly closely related species Gymnodinium maguelonnense.

Leishmania (Kinetoplastida): species typing with isoenzyme and PCR-RFLP from cutaneous leishmaniasis patients in Ethiopia.

Cutaneous leishmaniasis (CL) is an increasing public health problem in Ethiopia. There is a concern that it is spreading with increased incidence. In this study, we used isoenzyme electrophoresis and internal transcribed spacer one (ITS1) PCR-RFLP techniques to identify Leishmania species from CL patients in Ethiopia. We obtained isolates from 55 localized cutaneous leishmaniasis (LCL), 3 diffused cutaneous leishmaniasis (DCL) and 36 biopsy samples from 34 LCL and 2 DCL cases from All Africa Leprosy and Tuberculosis Rehabilitation and Training Center (ALERT) and clinically diagnosed CL cases from Ochollo village. Both isoenzyme and ITS1 PCR-RFLP techniques showed that Leishmania aethiopica (L. aethiopica) was the aetiologic agent in all cases. Our study also showed that ITS1 PCR-RFLP could identify Leishmania species from biopsy samples and suggests the method could be used for epidemiological surveillance of leishmaniasis in Ethiopia and for species-specific diagnosis.

Use of PCR for diagnosis of post-kala-azar dermal leishmaniasis.

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.