An evaluation of dark field microscopy, culture and commercial serological kits in the diagnosis of leptospirosis.

Context: This study was conducted to analyze the clinical utility of various leptospira diagnostic modalities. Aims: To evaluate the role of dark field microscopy (DFM), culture, immunochromatography (IgM Leptocheck), IgM enzymelinked immunosorbent assay (IgM ELISA), macroscopic slide agglutination test (MSAT) and microscopic agglutination test (MAT) in diagnosing leptospirosis in febrile patients. Settings and Design: Descriptive study conducted in a tertiary care hospital from January 2011 to April 2012. Subjects and Methods: Blood, urine and paired sera from 100 patients with clinical suspicion of leptospirosis (study group) were collected and subjected to DFM, culture, IgM Leptocheck, IgM ELISA and MSAT. Fifty randomly selected sera from febrile patients tested positive for infections other than leptospirosis (control sera) were also subjected to the aforementioned serological assays. All the leptospira seropositive samples were subjected to MAT. Statistical Analysis Used: Positive predictive values (PPV) and coefficient of agreement (kappa). Results: None of the clinical samples showed positivity by DFM. Leptospira inadai was isolated from a urine sample. The seropositivity of IgM Leptocheck, IgM ELISA and MSAT was 16%, 46% and 47%, respectively. The PPV of these assays was 14.3%, 8.7% and 6.5%, respectively. Poor agreement was obtained among these assays. Only four study group leptospira seropositive samples were confirmed by MAT with Australis being the predominant serovar. None of the leptospira-positive control sera were confirmed by MAT. Conclusions: DFM and culture have limited utility in diagnosing leptospirosis with serology being the mainstay. The present study shows discordant results with the commercially available serological kits. Further studies should be done to evaluate the various diagnostic modalities.

Dynamics of tear fluid desiccation on a glass surface: a contribution to tear quality assessment

BACKGROUND: Fern-like crystalloids form when a microvolume of tear is allowed to dry out at ambient conditions on a glass surface. Presence of crystalloids in tear "microdesiccates" is used to evaluate patients with Dry-Eye disease. This study aims to examine morphologically the desiccation process of normal tear fluid and to identify changes associated with accelerated tear evaporation. Tear microdesiccates from healthy (Non-Dry Eye) and Dry Eye subjects were produced at ambient conditions. Microdesiccate formation was monitored continuously by dark-field video microscopy. Additionally, accelerated desiccation of tear samples from healthy subjects was conducted under controlled experimental conditions. Particular morphological domains of tear microdesiccates and their progressive appearance during desiccation were compared. RESULTS: In normal tear microdesiccates, four distinctive morphological domains (zones I, II, III and transition band) were recognized. Stepwise formation of those domains is now described. Experimentally accelerated desiccation resulted in marked changes in some of those zones, particularly involving either disappearance or size reduction of fern-like crystalloids of zones II and III. Tear microdesiccates from Dry Eye subjects may also display those differences and be the expression of a more synchronous formation of microdesiccate domains. CONCLUSION: Morphological characteristics of tear microdesiccates can provide insights into the relative rate of tear evaporation.

Microdesiccates produced from normal human tears display four distinctive morphological components

Desiccation of human tears on glass surfaces results in fern-like crystalloids. This phenomenon has been associated with tear normality (Tear Ferning Test, TFT) and is used as a diagnostic aid to evaluate patients with Dry-Eye disease. However, TFT is focused on the assessment of only a minor fraction of desiccated tear samples and considers only the relative abundance and density of fern-like crystalloids. The aim of this study was to characterize morphologically entire desiccated micro volumes of tears from healthy donors. Tear samples were collected from 23 healthy young adult volunteers. Tear aliquots (1-3 μL) were allowed to dry on glass surfaces under ambient conditions of temperature (15-25°C) and relative humidity (40-45%). Dry samples were analyzed by dark-field microscopy. Morphometric data were acquired with Image J software. Tear volume was positively correlated with both area and time of desiccation. Morphological features of multiple microdesiccates produced from a single subject displayed striking similarities whereas tear microdesiccates from different healthy subjects displayed consistent differences but shared a common general design. This design may be mostly represented by the occurrence of four distinctive zones, named as zones I, II, III and Transition band. The main features of these zones are described.

Determination of presence of Tritrichomonas foetus in uterine lavages from cows with reproductive problems / Determinação da presença de Tritrichomonas foetus em lavados uterinos de vacas com problemas reprodutivos

The aim of this study was to determine the presence of Tritrichomonasfoetus in two dairy herds on the Altiplano Cundiboyacense. Twenty-one low-volume uterine lavages from cows with a history of reproductive problems in two dairy herds located in the municipality of Sibaté (Cundinamarca) and Ventaquemada (Boyacá) were evaluated. In the first herd, 10 cows were sampled and in the second, 11 cows, based on three inclusion criteria. The uterine lavages were obtained through infusion of physiological saline solution into the uterine body. The samples were centrifuged and seeded in Tritrichomonas basal medium for 10-15 days at 37 ºC. The protozoa were evaluated on the day of sampling and 10 and 15 days after incubation by means of direct viewing under a dark-field microscope. Positive samples were stained with Wright and Lugol to identify the morphological characteristics. This study showed that T. foetus was present in 61.8% of the animals sampled. The determination that T. foetus was present in 61.8% of the samples analyzed is a significant finding given that in the herds evaluated, this agent had not previously been diagnosed.

O objetivo do presente estudo, foi determinar a presença de Tritrichomonas foetus em dois rebanhos leiteiros no Altiplano Cundiboyacense. De vacas com um histórico de problemas reprodutivos, foram avaliados 21 lavados uterinos de pequeno volume, em dois rebanhos leiteiros localizados nos municípios de Sibaté (Cundinamarca) e Ventaquemada (Boyacá). No primeiro rebanho, 10 vacas foram amostradas e no segundo 11, baseado em três critérios de inclusão. Os lavados uterinos foram obtidos mediante infusão de solução salina fisiológica no corpo do útero. As amostras foram centrifugadas e semeadas em meio de cultura básico para Tritrichomonas por 10-15 dias a 37 ºC. Os protozoários foram avaliados no dia da colheita de amostras e 10 e 15 dias após incubação por meio de visualização direta sob um microscópio de campo escuro. As amostras positivas foram coradas com Wright e Lugol para identificar as características morfológicas. Este estudo mostrou que T. foetus estava presente em 61,8% dos animais amostrados. A determinação de que T. foetus estava presente em 61,8% das amostras analisadas é um achado significativo, dado que nos rebanhos avaliados, esse agente não tinha sido previamente diagnosticado.

Occurrence of leptospirosis among suspected cases in Chennai, Tamil Nadu.

Objective: To evaluate the serological profile of leptospirosis by microscopic agglutination test (MAT) and dark field microscopy (DFM) and to determine the serovar prevalence rate among patients with pyrexia of unknown origin. Materials and Methods: A total of 3830 blood samples were received from different hospitals and laboratories in and around Chennai. They were screened for leptospirosis by MAT and direct observation of live Leptospira by DFM. Results: A total of 748 (19.5%) Leptospira positive cases were identified; among these, 36.76% were Leptospira australis, 30% were Leptospira canicola, 14.57% were Leptospira autumnalis, 12% were Leptospira icterohaemorrhagiae, 4.68% were Leptospira patoc and 1.87% were Leptospira grippotyposa. Patients were in the age group of 1-86 years, with a median age of 43.5 years. 50% positive cases were in the age group of 10-35 years. Majority of the Leptospira infected cases were males (62.98%) than females (37.02%). Conclusion: Leptospirosis occurs in Chennai throughout the year although the number and positivity of cases increased during the monsoon season.