Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10-1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82-1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.

A Case Study of Anisakiasis in the Palatine Tonsils.

Anisakidosis is a nematode infection caused by the ingestion of larvae-infected raw or undercooked fish. Although gastric anisakiasis is a common disease in Japan due to the popularity of eating raw and undercooked fish, reports of anisakiasis in the tonsils are extremely rare. A 68-year-old man presenting with clinical features of peritonsillitis was admitted for examination. The right peritonsillar region exhibited slight edematous swelling and rash, and a white foreign body was observed. This foreign body was removed, and pathological examination revealed Anisakis.

Induction of protective immunity against Eimeria tenella infection using antigen-loaded dendritic cells (DC) and DC-derived exosomes.

Current methods for sustainable control of avian coccidiosis, whether by prophylactic medication or parasite vaccination, are suboptimal. In this study, we describe an alternative immunization strategy against Eimeria tenella infection using parasite antigen (Ag)-loaded dendritic cells (DCs), or their derived exosomes, in the absence of free Ag. CD45(+) intestinal DCs were isolated from E. tenella-infected chickens and loaded ex vivo with an extract of sporozoites as parasite Ag. Extracellular vesicles purified from the Ag-pulsed DCs expressed surface proteins associated with DC-derived exosomes, including major histocompatibility complex proteins (MHC I and MHC II), CD80, flotillin, and heat shock protein (HSP70). Following intramuscular immunization of chickens with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes, Ag-containing cells were observed diffusely localized in the lymphoid tissue and concentrated in germinal centers of caecal tonsils, and restricted to germinal centers (GC) in the spleen. Chickens immunized with pulsed DCs or exosomes exhibited (a) higher numbers of caecal tonsil and spleen cells expressing IgG and/or IgA antibodies that were reactive with E. tenella Ag, (b) greater numbers of IL-2-, IL-16-, and IFN-γ-producing cells, and (c) higher E. tenella Ag-driven cell proliferation, compared with chickens immunized with Ag in the absence of DCs or exosomes. Chickens immunized with Ag-pulsed DCs or Ag-pulsed DC-derived exosomes and subsequently given a live E. tenella challenge infection at 10d post-immunization displayed (a) increased body weight gains, (b) decreased feed conversion ratios, (c) reduced fecal oocyst shedding, (d) diminished intestinal lesions, and (e) lower mortality, compared with animals given Ag alone. This is the first demonstration of Ag-specific protective immunity against avian coccidiosis using parasite Ag-loaded DCs or DC-derived exosomes.

Viability and burden of Leishmania in extralesional sites during human dermal leishmaniasis.

BACKGROUND: The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients. METHODS: The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts. RESULTS: 7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates. CONCLUSION: Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis.

Changes of cytokines and IgG antibody in chickens vaccinated with DNA vaccines encoding Eimeria acervulina lactate dehydrogenase.

The aim of this study was to investigate the changes of cytokines and specific serum IgG in chickens following vaccination with DNA vaccines encoding either Eimeria acervulina (E. acervulina) lactate dehydrogenase (LDH) antigen or LDH and chicken IL-2 or IFN-γ. Two-week-old chickens were randomly divided into five groups. Experimental group of chickens were immunized with DNA vaccines while control group of chickens were injected with pVAX1 plasmid alone or sterile water. All immunizations were boosted 2 weeks later. The LDH-specific IgG antibody response was measured at weeks 1-6 post-second immunization. The result showed that the antibody titers in chickens vaccinated with DNA vaccines were significantly different from those of the control groups 1 week after the second immunization (P<0.05) and reached the maximum values 3 weeks post-second immunization. The systemic and local cytokine mRNA expression was determined by quantitative RT-PCR 7 days post-second immunization. The specific IgG antibody levels against LDH of all chickens vaccinated with vaccines were increased compared to those of sterile water (H(2)O) and plasmid (pVAX1) control chickens 1-6 weeks post-second immunization (P<0.05). The mRNA levels of IFN-γ, IL-2, TNFSF15, IL-17D as well as TGF-ß4 in both spleen and cecal tonsil were also increased in experimental chickens. In contrast, the only significant change of IL-4 mRNA level was observed in spleen of chickens immunized with pVAX-LDH-IL-2 compared with pVAX-LDH and control groups (P<0.05). These results suggested that DNA vaccines could increase the IgG antibody level and induce the expressions of cytokines.

Toxoplasma gondii inhibits R5 HIV-1 replication in human lymphoid tissues ex vivo.

Critical events of HIV-1 pathogenesis occur in lymphoid tissues where HIV-1 is typically accompanied by infections with other pathogens (HIV co-pathogens). Co-pathogens greatly affect the clinical course of the disease and the transmission of HIV. The apicomplexan parasite Toxoplasma gondii is a common HIV co-pathogen associated with AIDS development. Here, we examined the interaction of T. gondii and HIV in coinfected human lymphoid tissue ex vivo. Both pathogens readily replicate in ex vivo infected blocks of human tonsillar tissue. Surprisingly, we found that live T. gondii preferentially inhibits R5 HIV-1 replication in coinfected tissues. This effect is reproduced by treatment of the tissue blocks with recombinant C-18, a T. gondii-encoded cyclophilin that binds to CCR5. These ex vivo findings raise the possibility that, in addition to being a co-factor in HIV disease, T. gondii may influence the outcome of viral infection by preferentially suppressing R5 variants.

Leishmania infantum promotes replication of HIV type 1 in human lymphoid tissue cultured ex vivo by inducing secretion of the proinflammatory cytokines TNF-alpha and IL-1 alpha.

Parasitic infections such as leishmaniasis can modulate the life cycle of HIV-1 and disease progression. Coinfection with HIV-1 and Leishmania has emerged as a serious threat in countries where both pathogenic agents are widespread. Although there are numerous clinical reports illustrating the cofactor role played by Leishmania in HIV-1-infected patients, there is still no information on the contribution of Leishmania to the biology of HIV-1 in human lymphoid tissue that is considered a major in vivo site of virus production. In this study we explored the modulatory effect of Leishmania on the process of HIV-1 infection using ex vivo cultured human tonsillar tissue. We found that the protozoan parasite Leishmania enhances both HIV-1 transcription and virus production after infection of human tonsillar tissue infected ex vivo with viral strains bearing various coreceptor usage profiles. Studies conducted with pentoxifylline and neutralizing Abs revealed that the Leishmania-mediated increase in HIV-1 production was linked to a higher production of TNF-alpha and IL-1alpha. Our findings help to unravel the molecular mechanism(s) through which the two microorganisms interact and provide information that may be useful for the design of more effective therapeutic strategies aimed at controlling disease progression in persons dually infected with HIV-1 and Leishmania. This work also indicates that histocultures of human lymphoid tissue infected by both pathogens represent an ideal experimental cell system to dissect interactions occurring between HIV-1 and an opportunist pathogen in a human microenvironment that approximates conditions prevailing under physiological situations.

Experimental life cycle of Lagochilascaris major leiper, 1910 (Nematoda: Ascarididae) in cats (Felis domesticus).

The life cycle of Lagochilascaris major was studied using eggs collected from a natural clinical case in a domestic cat. Twenty-seven white mice (Mus musculaus), 5 hamsters (Mesocricetus auratus), and 1 vesper mouse (Calomys callosus) were orally inoculated with 800-1,300 embryonated eggs. When examined from 73 to 246 days postinoculation (PI), encysted third-stage larvae were seen in skeletal muscles and less frequently in connective tissue, liver, and lungs. Twenty-two of the 23 cats orally inoculated with 40-430 encysted larvae from these rodents, and necropsied from 1 hr to 185 days PI, became infected. Third-stage larvae were located in the stomach, esophagus, and oropharynx from 1 to 24 hr PI. At 48 hr, larvae, from mainly the fourth stage, were only found, unilaterally or bilaterally, inside a "sac" in the region of the semilunar fold of the palatine tonsil at the base of the tongue. Adult worms were found in this location from 10 to 175 days PI. No fistulated abscess to the outside medium was found. Adult worms were also found in the middle ears of 2 cats showing purulent otitis. Eggs in the ear secretion were under different stages of development. Eggs in feces were first observed on days 14 and 15 PI, and 1 cat shed them until 178 days PI. Six infected cats were treated with fenbendazole at 50 mg/kg of body weight for 3 consecutive days, eliminating all the parasites present in the tonsils. The drug was not effective against the parasites present in the middle ear. No stage of the parasite was found in the tissues of 5 cats given 4,000-5,200 eggs orally and examined after 19 and 50 days PI. This indicates that the life cycle of L. major requires an obligate paratenic host and is characterized by heteroxenic cycle.

[Malignant fibrohistiocytoma in otorhinolaryngology. Review of two cases]. / Fibrohistiocitoma maligno en ORL. Una revisión a propósito de dos casos.

Fibrohistiocytomas are soft-tissue tumors of histiocytic origin that have a variety of histological patterns. Although cases of malignant fibrohisticytoma (MFH) of the head and neck have been reported with increasing frequency in recent years, they can be considered rare. However, they probably are more frequent than thought because of the different designations that the disease has received and confusion among clinicians and pathologists regarding these tumors. We report two cases of MFH of the tonsil and larynx, respectively. The histopathological diagnostic criteria, therapeutic indications, and prognosis evaluation are described.

Anisakiasis of the tonsils.

A six-year-old Indian girl presenting with clinical features of chronic recurrent tonsillitis, had a tonsillectomy. Histopathology revealed a cross-section of an Anisakis worm in the tonsil. This report documents a new location in the human host for this rare parasite.

[Tonsillar hirudiniasis. Case report]. / Hirudiniasis amigdalar. A propósito de un caso.

Report on a tonsillar parasitosis in a 5-year-old child living in the countryside from leeches of Hirudinea. He consulted because a feeling of foreing body in the throat and spitting of blood, since a fortnight. The AA. remark the low incidence of those cases in our environment.

Oral leishmaniasis and Kaposi's sarcoma in an AIDS patient.

In the Mediterranean basin area, visceral leishmaniasis is an endemic disease caused by Leishmania donovani infantum. This study describes the clinical and pathological features of one patient with AIDS who had oral (tonsillar) leishmaniasis, caused by a viscerotropic zymodeme, concurrent with a Kaposi's sarcoma and with a CMV infection.

Leishmania donovani parasites in the nasal secretions, tonsillopharyngeal mucosa, and urine centrifugates of visceral leishmaniasis patients in Kenya.

In the early 1930s, investigators of visceral leishmaniasis stated that Leishman-Donovan bodies are found in body fluids of kala-azar patients, for example, in urine, feces, semen, and nasal and pharyngeal secretions. Based on this finding, we investigated the diagnostic potential of nasal secretions, tonsillopharyngeal mucosal swabs, and urine centrifugates inoculated into Schneider's Drosophila Medium (containing antibiotics and antifungal agents) as well as with Giemsa-stained smears. Consequently, 64 randomly selected patients with visceral leishmaniasis from Kenya (59 who were splenic culture or Giemsa stain positive and five who were culture negative but Giemsa stain positive) were tested by three noninvasive methods. These tests were all performed before the patients were treated with Pentostam. Cultures of nasal and tonsillopharyngeal swabs and urine centrifugates produced 28 positive samples representing 24 patients (37.5%). Moreover, a set of 25 Giemsa-stained slide smears made from the nasal and tonsillopharyngeal mucosa of 25 patients with visceral leishmaniasis who had not tested positive in cultures produced nine positives. Therefore, the overall total of patients who tested positive by all of the above methods was 33 or 51.6%. The cryopreserved Leishmania isolates were characterized by cellulose acetate electrophoresis using 20 enzyme systems. The isoenzyme profiles produced by the parasites were represented in five different L. donovani s.l. zymodemes. Representatives of these isolates were also characterized by DNA Southern blotting analysis, which corroborated the isoenzyme results.

Investigation into the occurrence of toxoplasma in pharyngeal and palatine tonsils by inoculation of tonsillar tissues into mice.

Among randomly chosen persons in the age group one to seven years the generally accepted proportion of positive dye tests for Toxoplasma is about eight per cent. The 124 children in this age group studied by us represent a selected group in which a higher percentage is to be expected. The group included 63 boys and 61 girls, from whom no blood samples were available for serological investigation. An arbitrary percentage of twice the eight per cent would result in 20 cases with a positive dye test. In the four successful attempts to isolate Toxoplasma all four cases were boys. Among 20 persons aged 14 to 21 years in whom a positive dye test could actually be established, one attempt to isolate the organism in a girl of 16 years of age was successful. No generalized lymph node enlargements were mentioned in the history of the five successful cases, only relapsing laryngo-pharyngeal symptoms. In the five successful isolations the Toxoplasma organisms present in the tonsillar organs appeared not to be virulent for the mice used; the organism was recovered in the cystic form from the brains of the mice. In subsequent animal passages the virulence could be influenced by the number of cysts per inoculum.