Heat fixed but unstained slide smears are infectious to laboratory staff.

Background and Aim: In a clinical microbiology laboratory, heat fixed slide smears are commonly transported from one place to another for staining with different stains and also for onsite proficiency testing of laboratory technicians for accreditation of the laboratories. These smears are frequently handled without gloves by the staff in developing countries. Therefore, this study was conducted to check the survivability of tubercle bacilli on smears after physical and chemical treatments. Methods: A total of 196 AFB positive smears were analyzed. Of these, 116 were stained with Ziehl Neelsen (ZN), 60 with cold Kinyoun and 10 were unstained but heat fixed and 10 were neither stained nor heat fixed. The last 20 smears served as controls. The ZN and Kinyoun stained smears were 0-1.5-year-old and stored at room temperature in slide boxes, while control smears were freshly prepared. All smears were prepared from sputum samples positive for acid fast bacilli. All four sets were subjected to slide culture to see if mycobacteria could survive and grow in any. For slide culture, a new and safe device was used, which is designed for three in one purpose: cell cultivation, direct observation of the growth under microscope and cell harvesting inside the closed tube. The slide smears were directly dipped into this tube that contained liquid culture medium. The tubes were incubated at 370C for four weeks. The growth, if any, was confirmed by MPT-64 rapid test and subculture on LJ slants. Results: No growth was observed in ZN and Kinyoun stained slide smears. However, significant growth was observed in both control sets; the unstained non heat fixed as well as heat fixed slide smears. Conclusions: The results of our study indicate that tubercle bacilli remain viable even after heat fixation and carry risk of infection by contact. However, stained smears are safe for handling and storage.

Comparison of Calcofl uor White M2R Fluorescence and Modifi ed Gram Chromotrope Kinyoun

Routine diagnosis of intestinal microsporidiosis in clinical diagnostic laboratories relies mostly on detection of microsporidial spores via special staining and microscopic techniques. This paper describes the comparative evaluation of Calcofl uor White M2R method, with modifi ed Gram-chromotrope Kinyoun method as the reference standard. One hundred and six stool samples were examined for the presence of microsporidial spores. Sensitivity, specifi city, positive and negative predictive values of the Calcofl uor White M2R method compared to the reference technique were 95.2%, 4.3%, 78.2% and 20.0%, respectively. The positive predictive value (PPV) was 78.2% and the negative predictive value (NPV) was 20.0%. Despite low specifi city of the CFW method due to its ability to stain chitinous wall of microorganisms, the presence of distinct deep-blue horizontal or equitorial stripes in microsporidial spores in modifi ed Gram-chromotrope Kinyoun would likely reduce the false positive results obtained in the Calcofl uor White M2R. Hence, the simultaneous use of these two methods would give better performance and accuracy for the detection of microsporidial spores in patients with intestinal microsporidiosis.

Técnicas para la detección de Cryptosporidium sp. en sistemas de tratamiento de agua residual / Techniques for Cryptosporidium sp. detection in wastewater treatment systems

En este trabajo se evaluó la aplicabilidad de la técnicas de tinción de Kinyoun e Inmunofluorescencia, para el monitoreo periódico de la calidad parasitológica del agua residual del Sistema de Lagunas de Estabilización "Planta de Tratamiento de Aguas Servidas Sur" de la ciudad de Maracaibo. Se detectó Cryptosporidium sp. en el 100 por ciento de las muestras analizadas por las dos técnicas. Los promedios de Cryptosporidium sp. detectados por la técnica de Kinyoun fueron: en la entrada de la planta 4,9x105 ooquistes/100L, en los módulos primarios 1,2x105 ooquistes/100L, en la laguna facultativas 5,9x104 ooquistes/100L y en la salida 3,0x104 ooquistes/100L, mientras que para C. parvum por inmunofluorescencia fueron: 5,9x104 ooquistes/100L, 7,7x104 ooquistes/100L, 3,0x105 ooquistes/100L y 3,0x104 ooquistes/100L, respectivamente. Se observó una correspondencia del 100 por ciento de positividad en las muestras analizadas por ambas técnicas. Dada las ventajas en facilidad de aplicación, rapidez y costos que ofrece la técnica de Kinyoun, se sugiere su aplicación como técnica de rutina para la evaluación de Cryptosporidium sp. en muestras de agua residual. Se observó la presencia de par sitos en el efluente final, por lo tanto éste no es apto desde el punto de vista parasitológico, para ser empleado con fines de irrigación, sin representar un riesgo para la salud pública.

This work evaluates the applicability of the Kinyoun and immunofluorescence techniques for periodic control of the parasitological quality of wastewater from the stabilization lagoon system “Wastewater Treatment Plant South” in the city of Maracaibo. Cryptosporidium sp. was detected in the 100 percent of the samples analyzed by both techniques. The concentration of Cryptosporidum sp. detected by the Kinyoun technique was: 4.9x105 oocysts/100L at the plant entrance, 1.2x105 oocysts/100L in the primary modules, 5.9x104 oocysts/100L in the facultative lagoon and 3.0x104 oocysts/100L in the final effluent, while those of C. parvum detected by inmunofluorescence were: 5.9x104 oocysts/100L, 7.7x104 oocysts/100L, 3.0x105 oocysts/100L and 3.0x104 oocysts/100L, respectively. A 100 percent correspondence of the results from samples analyzed by both techniques was observed. Given the advantages in ease of application, rapidity and costs offered by the Kinyoun technique, its routine application for evaluating Cryptosporidium sp. in wastewater samples is suggested. The presence of parasites in the final effluent was observed; therefore, from the parasitological viewpoint, it is not apt for irrigation purposes, without representing a risk to human health.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats / Detecção quantitativa de oocistos de Toxoplasma gondii, por um teste modificado de Kato Katz usando coloração de Kinyoun (KKK), em gatos infectados experimentalmente com a cepa ME49

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5x10² mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Detectamos oocistos de Toxoplasma gondii em fezes de gatos experimentalmente infectados, usando a abordagem de Kato Katz, com subseqüente coloração pelo método de Kinyoun. Animais sorologicamente negativos ao T. gondii foram infectados por via oral com 5x10² cistos da cepa ME49 de cérebros de camundongos. Fezes foram colhidas diariamente a partir do 3º até o 30º dia pós-infecção. Oocistos foram detectados por centrífugo-flutuação em sacarose qualitativa e pelo método quantitativo de Kato Katz modificado corado pela técnica de Kinyoun (KKK). Em gatos experimentalmente infectados, oocistos foram detectados do 7º ao 15º dia pela técnica de centrífugo-flutuação em sacarose, mas oocistos foram detectados do 6º ao 16º dia pelo KKK, sendo sensível por um período maior, com documentação permanente. O pico da excreção de oocistos ocorreu entre 8º a 11º dia pós-infecção, antes de resultado sorológico positivo. KKK pode ser utilizado na triagem e quantificação da excreção de oocistos em fezes de animais suspeitos, com redução da manipulação de material infectante, diminuindo a possibilidade de contaminação ambiental e do operador.

Demonstração de Isospora belli pelo método modificado de Kinyoun em paciente com a síndrome da imunodeficiência adquirida / Demonstration of Isospora belli bt modified Kinyoun's acid-fast stain in a patient with acquired immune deficiency syndrome

A Isopora belli como o Cryptosporidium parvum é um coccídio intestinal, encontrado em indivíduos imunodeficientes e imunologicamente normais. Este protozoário é facilmente identificado, quando corado pelos métodos derivados de Ziehl-Neelsen, usados para coloração do C. parvum e l. belli, em pacientes portadores da síndrome da imunodeficiência adquirida (SIDA/AIDS).