A corneal dystrophy associated with transforming growth factor beta-induced Gly623Asp mutation an amyloidogenic phenotype.

PURPOSE: To present the light and electron microscopic findings of a unique corneal dystrophy never before described in a German family carrying the Gly623Asp Mutation of the TGFBI gene with late clinical onset. DESIGN: Experimental study. PARTICIPANTS: Four affected and 6 nonaffected family members. METHODS: Slit-lamp examination, photographic documentation, and isolation of genomic DNA from peripheral blood leucocytes obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members. Five corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against keratoepithelin (KE) 2 and 15. MAIN OUTCOME MEASURES: Clinical and histologic characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene. RESULTS: The specimens showed destructive changes in Bowman's layer and the adjacent stroma. Patchy Congo red-positive amyloid deposits were found within the epithelium in 1 cornea, in Bowman's layer and in the anterior stroma of all specimens also showing KE2, but not KE15, immunostaining. Electron microscopy revealed deposits mainly located in the anterior stroma and Bowman's layer and in small amounts in the basal area of some epithelial cells. The destroyed areas were strongly Alcian blue-positive, the Masson Trichrome stain proved mainly negative for the deposits. All affected but none of the unaffected family members had a heterozygous missense mutation in exon 14 of the TGFBI gene (G-->A transition at nucleotide 1915) replacing glycin by aspartic acid amino acid (Gly623Asp) at position 623 of the KE protein. CONCLUSIONS: In contrast with the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al, our cases presented with Salzmann's nodular degeneration-like clinical features and their specimens contained KE2-positive amyloid. The reason for this now "meeting the expectation histologic phenotype" is unclear. The histologic findings emphasize that this is a unique corneal dystrophy, which shares no clinical characteristics with Reis-Bücklers' dystrophy and should be treated as a distinct entity. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Quantification of Trypanosoma cruzi in the heart, lymph nodes and liver of experimentally inffected mice, using limiting dilution analysis

Limiting dilution analysis was used to quantify Trypanosoma cruzi in the lymph nodes, liver and heart of Swiss and C57 B1/10 mice. The results showed that, in Swiss and B1/10 mice infected with T. cruzi Y strain, the number of parasites/mg of tissue increased during the course of the infection in both types of mice, although a grater number of parasites were observed in heart tissue from Swiss mice than from B1/10. With regard to liver tissue, it was observed that the parasite load in the initial phase of infection was higher than in heart. In experiments using T. cruzi Colombian strain, the parasite load in the heart of Swiss and B1/10 mice increased relatively slowly, although high levels of parasitization were nonetheless observable by the end of the infection. As for the liver and lymph nodes, the concentration of parasites was lower over the entire course of infection than in heart. Both strains thus maintained their characteristic tissue tropisms. The limiting dilution assay (LDA) proved to be an appropriate method for more precise quantification of T. cruzi, comparing favorably with other direct microscopic methods that only give approximate scores.

Delayed phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a fluorochrome assay: ultrastructural aspects

The few studies already published about phagocyte functions in Chediak-Higashi syndrome (CHS) has stated that neutrophils present slow rate of bacterial killing but normally ingest microorganisms. In the present study, both phagocytosis and killing of Staphylococcus aureus were verified to be in neutrophils from two patients with CHS when these functions were simultaneously evaluated by a fluorochrome phagocytosis assay. Electron microscopic examination showed morphologic differences among neutophils from CHS patients and normal neutrophils regarding the cytoplasmic structures and the aspects of the phagolysosomes. It was noteworthy the presence of giant phagolysosomes enclosing bacteria in active proliferation commonly observed in CHS neutrophils after 45 min of phagocytosis, wich corresponded with the impaired bactericidal activity of these leukocytes. The present results suggest that phagocytosis may also be defective in CHS, and point out to the sensitivity of the fluorochrome phagocytosis assay and its application in clinical laboratories.

New methods for the diagnosis of Babesia bigemina infection

Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currentlyused methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These testes have been used the fild but because they lack sensitivity and specificity, never and improved methods of diagnosis are being developed. The quantitative buffy coat (OBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with grater sensitivity and specificity than previously described serological tests. Similary, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of diferentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.

Morphological and virological studies in six autopsies of children with adenovirus pneumonia

Pulmonary lesions compatible with adenovirus infection were detected by gross and microscopic examination of autopsy tissues children aged from 5 to 34 months. Hepatic lesions indicative of systemic infection were also found in four of the chisldren. The viral etiology was confirmed in three cases by in-situ hibridization, electro-microscopy and immunofluorescence performed in parafin-embedded tissues, and in one case by cell culture isolation of adenovirus type 2 from nasopharyngeal exudate. Routine testing by methods additional to conventional light microscopy would probably have revealed a larger number of adenovirus infections among the 1.103 autopsy records analyzed in this study.

Altérations anatomopathologiques liées au traitement par cerclage des globes oculaires atteints de décollement de rétine [Histological changes related to scleral buckling for treatment of retinal detachment].

Treatment of retinal detachment frequently uses biocompatible materials to obtain scleral buckling. These materials are not devoid of consequences on surrounding tissues. In 3 eyes enucleated for failure of surgical treatment using scleral buckling materials, the changes prompted by episcleral implants could be observed. The sclera underwent both an inversion of its curvature and a reduction of its thickness under the material, as well as an encapsulation of the material was observed. While a silicone sponge was used in part to encircle one of these eyes, its capsular inner surface was regular and smooth. In contrast, hydrogel implants used in the three eyes showed a peripheral fragmentation prompting in two of them a typical foreign body giant cell granulomatous reaction. Changes in scleral curvature and scleral thinning were observed reflecting the consequences of the buckling procedure. The capsule formation occurred as it does for any nonabsorbable matérial implanted in tissues. Degradation and fragmentation of the hydrogel material suscitated a granuloma in response to fragments. These hydrogel specific changes should be recognized on microscopic examination of slides of either capsule or eyes previously in contact with this implanted material. They attested of the instability of hydrogel after implantation.

Chromoblastomycosis murine model and in vitro test to evaluate the sensitivity of Fonsecaea pedrosoi to ketoconazole, itraconazole and saperconazole

An experimental model of murine chromoblastomycosis and in vitro tests with Fonsecaea pedrosoi were used to test the sensitivity of this fungus to three different antimycotics. The experimental model was standardized in BALB/c mice inoculated intraperitoneally with a 10(6) CFU/ml suspension of a F. pedrosoi isolate. Clinical infection was evident after 5 days of inoculation. Three groups of 27 mice each were used in the experiment. One group was treated with ketoconazole (KTZ), another with itraconazole (ITZ) and the other with saperconazole (SPZ). Antimycotic therapy was continued for 21 days. The control group consisted of 40 mice which were inoculated, but not treated. Infection was documented by macroscopic and microscopic examination of affected tissue in addition to culture of tissue macerates. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) for the F. pedrosoi strain used were done. The in vitro results showed that SPZ was the most active with MIC 0.01 mg/ml and MFC 0.1 mg/ml, followed by ITZ. SPZ was also the most effective in vivo since 63% of the treated animals (p=0.01) showed a curative effect after the observation period. We concluded that SPZ had the best in vitro and in vivo activity against F. pedrosoi.

Experimental evaluation of an electromechanical artificial urinary sphincter in an animal model.

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The AMS 800 urinary control system is the gold standard for the treatment of urinary incontinence due to sphincter insufficiency. Despite excellent functional outcome and latest technological improvements, the revision rate remains significant. To overcome the shortcomings of the current device, we developed a modern electromechanical artificial urinary sphincter. The results demonstrated that this new sphincter is effective and well tolerated up to 3 months. This preliminary study represents a first step in the clinical application of novel technologies and an alternative compression mechanism to the urethra. OBJECTIVES: To evaluate the effectiveness in continence achievement of a new electromechanical artificial urinary sphincter (emAUS) in an animal model. To assess urethral response and animal general response to short-term and mid-term activation of the emAUS. MATERIALS AND METHODS: The principle of the emAUS is electromechanical induction of alternating compression of successive segments of the urethra by a series of cuffs activated by artificial muscles. Between February 2009 and May 2010 the emAUS was implanted in 17 sheep divided into three groups. The first phase aimed to measure bladder leak point pressure during the activation of the device. The second and third phases aimed to assess tissue response to the presence of the device after 2-9 weeks and after 3 months respectively. Histopathological and immunohistochemistry evaluation of the urethra was performed. RESULTS: Bladder leak point pressure was measured at levels between 1091 ± 30.6 cmH2 O and 1244.1 ± 99 cmH2 O (mean ± standard deviation) depending on the number of cuffs used. At gross examination, the explanted urethra showed no sign of infection, atrophy or stricture. On microscopic examination no significant difference in structure was found between urethral structure surrounded by a cuff and control urethra. In the peripheral tissues, the implanted material elicited a chronic foreign body reaction. Apart from one case, specimens did not show significant presence of lymphocytes, polymorphonuclear leucocytes, necrosis or cell degeneration. Immunohistochemistry confirmed the absence of macrophages in the samples. CONCLUSIONS: This animal study shows that the emAUS can provide continence. This new electronic controlled sequential alternating compression mechanism can avoid damage to urethral vascularity, at least up to 3 months after implantation. After this positive proof of concept, long-term studies are needed before clinical application could be considered.

Diagnostic performance characteristics of rapid dipstick test for Plasmodium vivax malaria

We compared the diagnostic performance characteristics of newly developed method, the rapid dipstick test, which provides colorimetric determination by developing antibody to the lactate dehydrogenase enzyme of parasites, with conventional standard thick-blood film examination. For the rapid test, OptiMAL commercial kits were used. The results were also evaluated with clinical findings from patients. The parasites were determined by microscopic examination of thick-blood films from 81 patients with vivax malaria from southeastern Anatolia, Turkey. The OptiMAL test results were found to be negative in five patients who were diagnosed clinically and through thick-film testing as having vivax malaria. There was no false positivity observed with the OptiMAL test. We concluded that this rapid malaria test has a lower level of sensitivity than the classical thick-blood-film test for malaria, but that these methods have equal specificity.

A simple modification of the Baermann method for diagnosis of strongyloidiasis

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37°C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.

Triatomines Involved in Domestic and Wild Trypanosoma cruzi Transmission in Concepción, Corrientes, Argentina

An entomological and serological survey was performed in three localities of the Department of Concepción, Province of Corrientes, Argentina in 1998 and 1999, to identify triatomines species involved in domestic and wild transmission of Chagas disease. Triatomines were collected by man/hour capture in 32 houses randomly selected and 44 nearby outdoor ecotopes. Trypanosoma cruzi infection in triatomines was assessed by direct microscopic observation (400x) of feces and polymerase chain reaction. Serological techniques used for people were Indirect Hemagglutination Test and Indirect Fluorescent Test. Triatomines were collected in 28.1% of the houses and 31.8% of the wild biotopes. Triatoma infestans (Klug 1834) was exclusively found indoors and T. cruzi infected 60% of them. Triatoma sordida (Stål 1859) was mainly found in extradomestic ecotopes where trypanosome infection rate reached 12.7%. Serological study of 98 local people showed that 29.6% were seroreactive; most of their houses were closed to wild biotopes colonized by T. sordida. Results indicate that there is an active T. infestans mediated transmission of Chagas disease in this zone that yields important human prevalence and that the populations of T. sordida in wild biotopes not only sustain the wild T. cruzi cycle but also represent an actual risk for people living in the area.

First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis). The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH) was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%), C. tropicalis (23%), C. glabrata (14%) and C. krusei (4%). This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR) amplification fragments for the precise tuberculosis (TB) diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture) test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients). Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

Infection rate of Babesia spp. sporokinetes in engorged Boophilus microplus from an area of enzootic stability in the State of Minas Gerais, Brazil

The infection rates of Babesia sporokinetes in engorged Boophilus microplus were evaluated during a 2-year period in a dairy farm located in an area of enzootic stability. Every 14 days engorged females were collected from calves and from adult animals. Ticks were incubated at 27 ± 0.5ºC and 80-90% relative humidity and Babesia infection rates were determined by microscopic examination of Giemsa-stained hemolymph smears. After 52 collections, 2105 ticks were obtained, from which 982 were collected from calves and 1123 from cows. The total Babesia infection rate was 10%, however the incidence was higher (p < 0.05) in ticks collected from calves (17.5%) than in those collected from cows (3.6%). Females collected from cows showed the highest infection rates in January, March, and August, and absence of infection in April and May. Ticks feeding on calves were infected throughout the experimental period. The infection rates of engorged females collected from naturally infected calves that were artificially infested with Babesia-free-larvae of B. microplus gradually decreased until the calves were four months old. No differences were observed among infection rates of ticks collected from calves maintained under natural conditions.

Evaluation of a PCR-RFLP assay for the identification of dermatophytes in situ

Dermatophytes are the main cause of superficial mycoses. These fungi have the capacity to invade keratinized tissue of humans or animals to produce infections that are generally restricted to the corneocytes of the skin, hair, and nails. Nevertheless, it is common to obtain negative results from fungal cultures of dermatological specimens where direct mycological examination showed fungal elements (30-40%). However, correct identification of the isolated dermatophytes from Tinea is important to choose the appropriate treatment. Therefore, we aim to develop a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA that is able to identify dermatophytes species in positive dermatological samples. PCR-RFLP identification of dermatophytes in skin or hair allowed validation of the results obtained in culture. It was also possible to identify the infectious dermatophytes when direct hair/ skin mycological examination showed fungal elements, but negative results were obtained from fungal culture. As a conclusion, PCR methods may provide significant benefits in the rapid diagnosis of Tinea. First, there is an increase in sensitivity of dermatophytes identification when enough material is available. Secondly, identification of the infecting agent can be obtained in 24 hours with PCR-RFLP or sequencing, whereas results from fungal cultures can take 2-3 weeks.