Balamuthia mandrillaris therapeutic mud bath in Jamaica.

Balamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission dynamics are poorly understood due to the high fatality rate and the sporadic nature of cases. Seventy-two soil samples were collected from beaches and the banks of lagoons, rivers, ponds, mineral springs and streams from across Jamaica and assayed for the presence of B. mandrillaris. Seventy-nine sites were sampled and the mitochondrial 16S rDNA gene of B. mandrillaris was amplified and sequenced to confirm the presence of the amoeba. One isolate of B. mandrillaris was recovered from soil from mineral spring which hosts an informal therapeutic mud bath business. Although B. mandrillaris is less frequently isolated from soil than other free-living amoebae, rubbing mud containing the organism onto the skin increases the likelihood of exposure and infection. This first report on the isolation of B. mandrillaris in the Caribbean and its presence in soil where human contact is likely warrants further investigation using serological methods to elucidate exposure patterns.

Evaluation of the in vitro activity of commercially available moxifloxacin and voriconazole eye-drops against clinical strains of Acanthamoeba.

PURPOSE: Acanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as "Acanthamoeba keratitis" (AK) and, more rarely, an infection of the central nervous system called "granulomatous amoebic encephalitis" (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. Moxifloxacin, a fourth generation fluoroquinolone, has been used with other drugs to treat GAE, but its efficacy as a treatment for AK is not known. Voriconazole has been used to treat AK; however, its cysticidal efficacy is not known. Both drugs are commercially available as eye-drops. The aim of this study was to evaluate the in-vitro activity of these eye-drops against Acanthamoeba compared to two reference drugs (chlorhexidine and amphotericin B) which are currently used to treat AK and GAE. METHODS: The sensitivity of two clinical and one type strain of Acanthamoeba to the commercial concentrations of the four drugs was evaluated with a colorimetric assay. Mature cysts were incubated with voriconazole to determine their sensitivity to this drug. The effects on cell proliferation and cell toxicity were determined using standard procedures with commercial kits. RESULTS: The four compounds were active against the Acanthamoeba strains in this study. Although it prevented encystation, moxifloxacin's amoebicidal activity was low. Voriconazole activity was greater than that of the other drugs, even at a concentration lower than in commercial eye drops. It was effective against cysts and decreased cell proliferation, with low cellular cytotoxicity. CONCLUSION: Voriconazole could be used against AK as a first-line treatment or in combination. Moxifloxacin is an interesting adjuvant to consider as it is effectively prevents encystation of the amoeba which often complicates infection resolution. In addition, moxifloxacin is effective in preventing secondary bacterial infections.

Galectin and aldolase-like molecules are responsible for the specific IgE response in humans exposed to Dirofilaria immitis.

Dirofilaria immitis is the agent of the heartworm disease in canids and felids, and of pulmonary dirofilariosis in man. Like other filariae, D. immitis harbours endosymbion Wolbachia bacteriae. In this work we analyse the response of specific IgE antibodies against both D. immitis antigens and the Wolbachia surface protein (WSP) in two groups of persons living in an area of canine endemia, one presenting high levels of total IgE (group 1) and other with normal levels (group 2). Infections with D. immitis were demonstrated by the presence of specific IgG in 228 individuals(48.8%) of the group 1 and only in one of the group 2. Specific IgE antibody response against D. immitis antigens was detected only in individuals of the group 1. IgE response against WSP was not detected in any group. The IgE response was directed mainly against two molecules of 33 and 42 kDa of the antigenic extract of D. immitis. These molecules were identified by mass spectrometry as a galectin and an aldolase, respectively. Their possible role in the survival mechanisms of the parasite and their contribution to development of allergic reactions in individuals resident in areas with heartworm disease are discussed.

Concurrent chemoradiation for locally advanced stage III non-small cell lung cancer with cisplatin, vinorelbine, and thoracic radiotherapy: a phase II study from the Galician Lung Cancer Group.

PURPOSE: The aim of this phase II study was to evaluate the activity and safety of the combination of cisplatin and vinorelbine with thoracic radiotherapy in unresectable locally advanced stage III non-small cell lung cancer (NSCLC). The primary endpoint was the objective response rate (ORR). Secondary objectives included toxicity profile, progression-free survival (PFS), and overall survival (OS). MATERIALS AND METHODS: A total of 48 NSCLC patients were enrolled (median age 60 years, 52% stage IIIA and 48% stage IIIB, 52% adenocarcinoma). Patients received three cycles of chemotherapy every 21 days [intravenous cisplatin 80 mg/m2 and intravenous vinorelbine 25 mg/m2 on day 1 and oral vinorelbine on day 8 (60 mg/m2)] concurrent with radiotherapy (66 Gy, administered at 1.8 Gy per day, five consecutive days per week). RESULTS: ORR was 79.2% (72.9% showing partial response and 6.3% showing complete response). With a median follow-up of 20.7 months, median PFS was 12 months and median OS was 36 months. Grade 3/4 toxicities were: neutropenia (14.5%), anaemia (6.2%), vomiting (2%), and oesophagitis (4.2%). No toxic deaths were reported. CONCLUSION: This combined regimen shows efficacy and a manageable safety profile. PFS and OS outcomes are encouraging and warrant further research.

Isolation of Naegleria fowleri from a domestic water tank associated with a fatal encephalitis in a 4 month-old Venezuelan child.

This study describes the association of household water system contamination with the pathogenic Free-Living Amoeba (FLA) Naegleria fowleri and a case of fatal Primary Amoebic Meningoencephalitis (PAM) in a child from the state of Monagas in Venezuela. Amoebae were initially identified by microscopy from a sample of cerebrospinal fluid (CSF) from the child. Direct DNA extraction and specific PCR/sequencing for N. fowleri was also carried out from the same CSF sample. In order to determine a possible environmental source of infection, water samples from the water tank of the child's home and also water bodies recently visited by the child and his family, were examined for the presence of N. fowleri by culture and PCR/sequencing. The results obtained from the collected water samples revealed that only the water tank of the house was positive for N. fowleri. PCR/sequencing showed that the strains isolated from the patient and the water tanks were 100 % identical. Therefore, the house water tank was confirmed as the source of infection in this case, possibly as a result of the occasional immersion of the child´s head under the water while bathing. This case highlights a novel source of thermally polluted water and another threat of N. fowleri infection.

The sera from individuals suffering from cutaneous leishmaniasis due to Leishmania brazilensis present antibodies against parasitic conserved proteins, but not their human counterparts.

Sera from individuals suffering from leishmaniasis have been shown to strongly react against conserved proteins from the parasite, such as ribosomal, histones and heat-shock proteins. Some of these proteins have also been described as immunogenic in several auto-immune syndromes, and the detection of antibodies against them is considered to be indicative of disorder of the immune system. In this paper, we investigate whether there is any relationship between the recognition of some conserved proteins from leishmania braziliensis by individuals suffering from cutaneous (CL) and mucocutaneous (MCL) leishmaniasis, and the recognition of the human homologues of these antigens found in sufferers from autoimmune diseases. Our findings reveal that the immune response generated during CL and MCL is elicited specifically by the parasitic histone H1 and Hsp70, since the CL and MCL sera do not react against their human counterparts. In addition, evidence is presented showing the specific recognition of human proteins by the autoimmune sera, showing only a weak cross-reaction with the most divergent regions of the parasitic proteins.

A specific primer pair for the diagnosis and identification of Acanthamoeba astronyxis by random amplified polymorphic DNA-polymerase chain reaction.

Random amplified polymorphic DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that is useful for species identification. In this study, a distinctive 485-bp band in Acanthamoeba astronyxis band patterns was found, using the OPC20 primer (ACTTCGCCAC). The band specificity was confirmed by hybridization, using it as a probe, against all OPC20 amplifications from different Acanthamoeba species. Once the fragment was sequenced, we used it to design a specific primer pair that was useful for the identification of different isolates as A. astronyxis species.

A serologic study of human toxocariasis in the Canary Islands (Spain): environmental influences.

The presence of anti-Toxocara antibodies in the human population of the Canarian Archipelago was studied by means of an indirect enzyme-linked immunosorbent assay with excretory/secretory antigen of infective-stage larva. A total of 14,074 were analyzed, resulting in the following distribution and prevalence by location: southern Tenerife 6,435 (2.5% positive), northern Tenerife 1,710 (6.7% positive), La Palma 1,214 (4.5% positive), La Gomera 264 (6.8% positive), El Hierro 204 (3.9% positive), Gran Canaria 2,875 (2.3% positive). Fuerteventura 277 (1.8% positive), and Lanzarote 1,095 (2.9% positive). The weighted average of the entire Archipelago was 3.4% positive. Neither age nor sex was found to be significantly associated with positive serology. The influence of the different Canarian mesoclimates on the spreading of this zoonosis is demonstrated, with significant differences being found between humid and arid islands. Likewise, a significant positive correlation was found between De Martonne's humidity-aridity index and percentage of people with evidence of parasite exposure on the different islands.

PCR-ELISA for diagnosis of mucocutaneous leishmaniasis.

In this work we demonstrate that the PCR-ELISA technique is sufficiently sensitive and specific for use as a diagnostic test in cases of mucocutaneous leishmaniasis. DNA was extracted from cultures of Leishmania braziliensis, Leishmania infantum, Leishmania tropica, Leishmania mexicana, Trypanosoma cruzi, and blood samples from individuals who presented a clinical diagnosis of leishmaniasis as well as from healthy individuals. The DNA was PCR amplified and the product obtained was hybridised with a biotin-labelled probe, the sequence of which was designed in our laboratory. The result of the hybridisation was visualised by means of an ELISA technique using antifluorescein antibody labelled with alkaline phosphatase and p-nitrophenylphosphate (pNFF) as chromogen. The optical density of the products of the pNFF hydrolysis was quantified in a spectrophotometer at a wavelength of 405 nm. Using this technique the percentage of detection was 83.3% in blood samples from patients clinically diagnosed as having mucocutaneous leishmaniasis. No false positive results were obtained.

A subtropical case of human babesiosis.

This report constitutes the first well-documented case of symptomatic human babesiosis from a subtropical site, south of the 40th parallel. This paper describes the definitive identification of Babesia divergens infection in a splenectomized patient from the Canary Islands.

Molecular characterization of the Leishmania braziliensis L6 ribosomal protein.

By screening a Leishmania braziliensis complementary DNA library with a pool of sera from leishmaniasis patients, the gene coding for L6 ribosomal protein was isolated. The sequence, genomic organization, and transcription of this gene are described in this article. The sequence analysis of the L. braziliensis L6 gene shows a single open reading frame, which codes for a protein of 192 amino acids (aa) with a hypothetical molecular mass of 20.9 kDa. The protein exhibits significant sequence similarity to L6 ribosomal proteins from higher eukaryotes and yeast. Thus, the L. braziliensis L6 protein contains 4 functional motifs, which are located at equivalent positions in other L6 ribosomal proteins described previously. Interestingly, the L6 ribosomal protein from L. braziliensis contains a specific region of 14 aa and a tyrosine kinase motif, which is absent in human and C. elegans L6 protein. The locus coding the L. braziliensis L6 ribosomal protein is formed by 2 gene copies arranged in tandem and located in a chromosome of approximately 0.9. Mb. The genes are actively transcribed as 2 polyadenylated transcripts of approximately 1.15 and 0.85 kb, which differ in their steady-state level and stability.

Helminths of the wild rabbit (Oryctolagus cuniculus) in Macaronesia.

Two hundred and four rabbits from 8 Macaronesian islands (Pico, San Jorge, San Miguel, Terceira, and Flores from Azores Archipelago; Tenerife and Alegranza from Canary Islands; and Madeira from Madeira Archipelago) were examined for helminth parasites between 1995 and 2000. Three species of cestodes, Taenia pisiformis (larvae), Andrya cuniculi, and Mosgovoyia ctenoides, and 5 species of nematodes, Trichuris leporis, Graphidium strigosum, Trichostrongylus retortaeformis, Passalurus ambiguus, and Dermatoxys hispaniensis, were identified. Only 3 species (M. ctenoides, T. retortaeformis, and P. ambiguus) were regularly distributed over the 3 archipelagos. Taenia pisiformis was not collected in Madeira, nor was A. cuniculi in the Azores and G. strigosum in the Canary Islands. Trichuris leporis and D. hispaniensis were only found in Madeira. Significant differences in the general prevalence of the nematodes G. strigosum and T. retortaeformis were detected between Azores and Madeira. The prevalence of T. retortaeformis differs significantly between the Azores and the Canaries and that of P. ambiguus was higher in Madeira than in Azores and Canaries. The helminth richness found in the wild rabbit in these Macaronesian archipelagos was very low compared with the Palearctic helminth fauna of this host. The wild rabbit was introduced from the Iberian Peninsula into different Macaronesian islands. Helminths introduced with Oryctolagus cuniculus into these islands also are commonly found in Iberian wild rabbits, which are excellent colonizers, as demonstrated in this study.

Validation of a rapid method for extraction of total RNA applied to Leishmania promastigotes.

This report explains a rapid procedure (approximately 50 min) for the isolation of highly purified total RNA from Leishmania promastigotes based on extraction with acidic phenol. The simplicity of the manipulations required make this method ideal for processing multiple samples; the quality of the RNA obtained is suitable for reverse transcription polymerase chain reaction analysis.

Cloning and molecular characterization of the cDNA encoding histone H1 from Leishmania braziliensis.

The isolation and molecular characterization of the histone H1-encoding gene from Leishmania braziliensis was carried out. The gene is present in the genome as a single copy and transcribed as a polyadenylated transcript of 830 nucleotides. The deduced amino acid sequence has in its central region the DNA binding K-[K/R]-A-A-[A/P] motif, which is repeated in tandem 9 times.

Small-scale isolation of high molecular weight DNA from Leishmania braziliensis.

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.

Catecholamines, potassium, and beta-blockade.

Stress-induced hypokalemia represents a transient risk factor for malignant arrhythmias in patients with coronary artery disease. The adrenergic nervous system serves as a major control system for the transport of potassium across the cell membrane. Nonselective beta-blockade presents epinephrine-mediated hypokalemia. This helps explain a possible mechanism for the significant improvement in morbidity and mortality rates of patients taking long-term propranolol therapy in the Beta Blocker Heart Attack Trial report.

Platelet aggregation and atherosclerosis.

The relationship of platelet aggregation to coronary and cerebral ischemic attacks has been well established. A number of clinical trials have shown that the daily use of aspirin 160 to 325 mg daily, dipyridamole 300 to 400 mg daily, or both are effective in preventing platelet aggregation, and results in a statistically significantly reduction in the incidence of coronary events and transient ischemic cerebral attacks.

Wide complex tachycardia: diagnosis and treatment.

A wide QRS complex tachycardia that cannot be immediately identified, and which on clinical grounds requires prompt treatment, should be converted electrically. Drug management can be time consuming, and agents that block AV transmission, that is, verapamil and propranolol, may be life-threatening.

The Miami Vices in the CCU. Part I. Cardiac manifestations of cocaine use.

There is a widespread misconception that the social use of cocaine is not associated with serious medical complications. It is therefore necessary to reemphasize that cocaine use can precipitate life-threatening cardiac events even in the absence of underlying heart disease.

Use of the "diving reflex" in paroxysmal atrial tachycardia.

Immersing the patient's face in cold water to induce the "diving reflex" is a quick and simple method of treating paroxysmal supraventricular tachycardia. Patients can be trained to perform the "diving reflex" at home. There are many advantages to this method of treatment when compared to many of the other therapies. Polyuria up to 3 L is often noted during the first 90 minutes of a paroxysmal tachycardia. The diuresis is thought to be due to atrial distention with resultant inhibition of antidiuretic hormone secretion.