Improved sensitivity of direct microscopy for Mycobacterium tuberculosis in lymph nodes: comparison of fluorescent microscopy and modified bleach method ZN microscopy along with conventional ZN staining method

Background: Extra pulmonary tuberculosis arises as a result of lymphatic spread from a primary focus. Fine needle aspiration cytology has assumed an important role in the evaluation of peripheral lymphadenopathy as a possible minimally invasive alternative to excisional biopsy. In most low-income countries; the only practically available bacteriologic method for diagnosing EPTB is direct smear microscopy for acid fast bacilli from the sample of the lesion. There are various methods of staining and concentration for improving sensitivity of direct microscopy for detection of tubercle bacilli in specimen.Methods: This prospective study was carried out in the Department of Pathology, Subharti Medical College and associated Chattrapati Shivaji Subharti Hospital, Meerut for a period of 2 years from July 2016 - August 2018 in 151 patients with clinical suspicion of TB and significant lymphadenopathy.Results: AFB positivity increased from 40.39% on conventional ZN stain to 48.34% on modified bleach method ZN stain and to 56.29% on Auramine-O fluorescent stain. Taking fluorescent microscopy (Auramine-O) as reference method the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of conventional ZN stain and modified bleach method ZN stain were calculated as 71.8%, 100%, 100%, 73.33%, 84.10% and 85.33%,100%,100% ,84.61% ,92.05%, respectively.Conclusions: The addition of fluorescent microscopy (Auramine-O) and modified bleach method ZN microscopy along with conventional ZN staining method would be an important adjunct to improve the microscopic detection of Mycobacterium tuberculosis in fine-needle aspirates of lymph nodes.

Risk factors and infection due to Cryptosporidium spp. in dogs and cats in southern Rio Grande do Sul / Fatores de risco e infecção por Cryptosporidium spp. em cães e gatos no sul do Rio Grande do Sul

Abstract This study investigated the frequency of oocysts of Cryptosporidium spp. in feces from dogs and cats in five municipalities in the southern region of the state of Rio Grande do Sul. The risk factors associated with infection were also investigated. Feces samples from 110 dogs and 18 cats were stained using the auramine method. At the time of feces sampling, a questionnaire with semi-open-ended questions was applied to the animal guardians and all data obtained underwent statistical analysis. The real frequency of oocysts of Cryptosporidium spp. was 24.63% (27 dogs and two cats). Only four samples of dog feces were diarrheic and no presence of oocysts was observed in any of them. Variables that represented risk factors for infection were: homemade food, untreated water, circulation of animals on grassy terrain and living in the same environment as other animals (cattle). The results made it possible to inferring that within the population studied, the frequency of parasitism due to Cryptosporidium spp. in dogs was relevant and emphasize the asymptomatic nature of this infection. The adopting control measures are highlighted, particularly in relation to variables that represent risk factors for this infection.

Resumo Este estudo verificou a frequência de oocistos de Cryptosporidium spp. em fezes de cães e gatos em cinco municípios da região sul do Rio Grande do Sul e fatores de risco associados à infecção. Amostras de fezes de 110 cães e 18 gatos foram coradas pelo método de auramina. No momento da coleta de fezes aplicou-se um questionário aos tutores dos animais com questões semiabertas e os dados foram submetidos à análise estatística. A frequência verdadeira de oocistos de Cryptosporidium spp. foi de 24,63% (27 cães e 2 gatos). Apenas quatro amostras de fezes caninas eram diarreicas e todas sem oocistos. As variáveis que representaram fatores de risco para a infecção foram: alimentos de preparo caseiro, água não tratada, circulação dos animais em terreno gramíneo e convivência com outros animais, principalmente bovinos. Os resultados sugerem que a frequência de cães parasitados por Cryptosporidium spp. é relevante, reforçando o caráter assintomático da infeção. Destaca-se a importância da adoção de medidas de controle, particularmente das variáveis que representaram fatores de risco à infecção.

Leaf histology of greenhouse plants, in vitro cultured and somaticembryo-derived plants of Bactris gasipaes Kunth / Histologia de folhas de Bactris gasipaes Kunth de plantas de casa-de-vegetação, cultivadasin vitro e obtidas por embriogênese somática

In vitro cultures of peach palm (Bactris gasipaes Kunth) were established by somatic embryogenesis but some improvements in maturation and conversion steps are still needed. The aim of this study was to analyze morpho-anatomical differences in peach palm leaves from greenhouse cultured plants, in vitro plants developed from in vitro germinated seeds and somatic embryo-derived plants. Expanded leaves were prepared for histological analyses and scanning electron microscopy. No significant difference was found between ex vitro and in vitro cultured plants, but the somatic embryo-derived plants showed structural alterations of the leaves. The epidermal cells were elongated in shape, the mesophyll cells were thicker and the vascular bundle was not very developed. In somatic embryo-derived leaves the cuticle was thinner than in other leaves and epicuticular wax was present but poorly deposited. In in vitro cultured plants, the deposition of epicuticular wax on the leaves was irregular while in the greenhouse plants it was regular and abundant. These alterations in somatic embryo-derived leaves could hinder the acclimatization and development of peach palm plants so it is necessary to improve the protocol for somatic embryogenesis to produce better plants.

O cultivo de pupunha (Bactris gasipaes Kunth) in vitro foi estabelecido através de embriogênese somática; alguns melhoramentos nas fases de maturação e conversão, contudo, ainda são necessários. O objetivo deste trabalho foi analisar diferenças morfoanatômicas em suas folhas, cultivadas em casa de vegetação, germinadas in vitro e provenientes de embriogênese somática. Folhas expandidas foram preparadas para análise histológica e microscopia eletrônica. Houve diferenças significativas entre as plantas da casa de vegetação e as plantas obtidas por embriogênese somática. As células epidérmicas eram alongadas; a espessura da folha e do clorênquima era menor que nas outras; a cutícula era menos espessa, com baixa deposição de ceras. Os feixes vasculares estavam menos desenvolvidos. As folhas das plantas cultivadas in vitro e de embriogênese somática apresentavam estruturas pouco desenvolvidas, o que sugere a necessidade de uma melhoria na fase de conversão durante a embriogênese somática.

Observation and Application of Oral Ambroxol in Improving Positive Rate of Tuberculosis Laboratory Test by Sputum Smear / 现代检验医学杂志

Objective To research the application of oral ambroxol in improving positive rate of sputum smear laboratory testing of Tuberculosis.Methods After immediate microscopic phlegm examination on 1 536 tuberculosis outpatients,them were divided into two groups alternatively and randomly.The referential group took oral placebo while the test group took oral ambroxol.Recheck would be conducted onmorning phlegm after three days,then comprated the positive rate of each group.Results The positive rates of immediate microscopic phlegm examination of the referential and test groups were 4.82％ and 4.56％ (x2 =0.058,P＞0.05) respectively.For the referential group,the positive rates before and after taking medicine were 4.82％ and 5.60％ respectively (x2 =0.475,P＞0.05).The differences had no statistic meaning.The positive rates of morning microscopic phlegm examination after taking medicine of the referential and test groups were 5.60％ and 8.59 ％ ()x2 =5.224,P＜0.05) respectively.The positive rates of the test group before and after taking medicine were 4.56 ％ and 8.59％ respectively (x2 =10.18,P＜0.05).It could be deemed that the difference had statistic meaning.Conclusion O-ral ambroxol can help raise the positive rate of microscopic phlegm examination of the patient with suspected tuberculosis.

TLC-SERS for detection of the dyed Croci Stigma / 药学实践杂志

Objective To establish a thin layer chromatography-surface-enhanced Raman scattering(TLC-SERS) method for the detection of dyed Croci Stigma.Method The dyed Croci Stigma was wetted by ethanol and pressed on TLC plate sprayed with Silver sol.The pressed zone was immediately detected by a portable Raman spectrometer.Factors including the concentration of ethanol, spraying time and amount of silver sol were investigated and optimized.Results The dyed Croci Stigma, stained by auramine O, new fuchsin, tartrazine and brilliant ponceau were successfully detected using the established method.Conclusion The combination of TLC and SERS technology provides a rapid, simple and sensitive detection method which is suitable for quick field tests.

Rapid detection of Chinese herbal medicine dyed by auramine O, amaranth and sunset yellow by ion mobility spectrometry / 中国药学杂志

OBJECTIVE: To develop a fast detecting method for three dyes (auramine O, amaranth and sunset yellow) illegally added into Chinese herbal medicine by ion mobility spectrometry (IMS). METHODS: The analysis was performed on ion mobility spectrometry, with source voltage 1 800 V for negative source and 2 300 V for positive source, drift tube voltage 7 500 V, gas inlet temperature 180℃, drift tube temperature 180℃, gate voltage 45 V, gate pulse width 120 μs, drift gas flow 1.2 L·min-1, exhaust pump 0.8 L·min-1, run time 30 s and spectrum length 25 ms. The samples were extracted by methanol, and then injected into the IMS system. The judgement of whether dye was added or not was made by comparing the migration time of the test samples with that of the reference substances. RESULTS: The auramine O, amaranth and sunset yellow could be rapidly identified. The minimum detection concentration of each compound was determined according to the signal to noise ratio (S/N) of 3. CONCLUSION: The IMS method for detecting auramine O, amaranth and sunset yellow illegally added into Chinese herbal medicine is simple, rapid and expected to be used as an initial screening method in drug rapid detecting system.

Determination of seven yellow pigments illegally added into Chinese materia medica by HPLC-MS / 中草药

Objective: To establish a rapid method for the simultaneous determination of seven yellow pigments (tartrazine, sunset yellow, brilliant yellow, orange II, auramine O, astrazon orange G, and basic orange) which were illegally added into Chinese materia medica (CMM). Methods: The sample was extracted with ultrasonic by 70% ethanol, separated on a reversed phase C18 chromatographic column by gradient elution using a mobile phase made up of acetonitrile and 0.05 mol/L ammonium acetate solution, and then detected by diode array detector in the wavelength of 432 nm and mass spectrometry detector with ESI+ ion source. Results: Under the condition of the above chromatography and mass spectrometry, the separating degree, specificity, and durability of the seven yellow pigments were good. RSD of precision was between 0.4%-1.8%. Above pigment solution was stable in 24 h and RSD < 1.9%. The recovery rates of accuracy were between 95.1%-102.9%. The limits of detection (LODs) were in the range of 2-31 μg/kg, and the RSD of repeatability was in the range of 0.3%-1.4%. Auramine O was detected in three batchs of CMM for sale. Conclusion: This method is simple, rapid, reproducible and has high sensitivity, which can be used for the determination of seven yellow pigments illegally added into dyed CMM, and it can provide the reference or help for relative company and State Food and Drug Administration in controlling the quality of CMM.

Screening of Auramine O in Guci Tablets by HPLC / 中国药师

Objective:To screen auramine O in Guci tablets. Methods:Auramine O was detected by HPLC method on a Diamon-sil-C18(250mm×4.6mm,5μm)columnwiththemobilephaseofacetonitrile-0.025mol·L-1monopotassiumphosphatebuffersolu-tion(containing 0. 2% triethylamine,adjusting pH to 3. 0 with phosphoric acid)(36∶65)at the flow rate of 0. 8 ml·min-1. The detec-tion wavelength was 432 nm and the column temperature was 35℃. Results:The LOD of auramine O was 0. 05ng. Among 45 batches of tested samples, auramine O was found in seven ones. Conclusion:The method is simple, rapid and accurate, which can guarantee the drug safety of Guci tablets.

Comparative analysis for the detection of tuberculosis by conventional and flurosence microscopy.

Total of 300 TB patients sample were collected from NIDCH (Indoor and Outdoor) for six months period where three different specimens (sputum, pus, BAL) were collected among different age group (male and female). In sputum sample, percentage of identified samples in Auramine-O stain was 41% whereas in Z-N stain 32%; in pus sample, Auramine stain is 22% and Z-N stain 15%; In BAL sample, Auramine stain is 12% and Z-N stain is 10%. Therefore, out of 300 samples, 25% of positive samples were identified by Auramine-O staining whereas 19% of positive samples were identified by Z-N stain. In all the cases Auramine-O is found better for identification of TB than Z-N. In sputum, male positive 10%, female positive 3.66%; in pus sample, male positive 5% and female positive 2.33%; in BAL sample, male positive 3% and female positive 1%. Age group of 26 to 45 years were found high rate of TB in male patients.

Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa.

Purpose: The study was conducted to compare different methods of detection of pathogenic protozoan parasites in stool specimens of People Living with HIV/AIDS (PLHA). Materials and Methods: Stool specimens of 242 HIV sero-positive patients were examined using the wet mount technique, modified Ziehl-Neelsen's (ZN) staining, auto-fluorescence and auramine fluorescence staining. Patient specimens, 94 and 40 out of 242, were also subjected to Giardia antigen detection using an enzyme immunoassay and Cryptosporidium antigen detection by immuno-chromatography, respectively. For calculation of sensitivity, specificity, positive and negative predictive values, light microscopy of wet mounts and modified ZN stained smears for Giardia and Coccidia, respectively, were considered as gold standards. Results: Sensitivity of auto-fluorescence, auramine-O staining and antigen detection techniques was found to be 100% as compared to the routine standards. The specificity of auto-fluorescence was 90.6% and 100% for Cyclospora and Isospora, respectively; that of auramine-O staining was 98.9% for Cryptosporidium, 99.30% for Cyclospora and 100% for Isospora; and that of antigen detection was 90.6% and 97.7% for Cryptosporidium and Giardia, respectively. Conclusion: In laboratories requiring screening of large number of stool specimens for detection of protozoan parasites, fluorescence microscopy and antigen detection can be useful techniques. Confirmation of positive results, however, needs to be done with the standard techniques.

Use of fluorescein diacetate (FDA) staining to detect viable Mycobacterium tuberculosis.

Objective: Drug resistant tuberculosis has long been a common problem prevailing in developing countries including Bangladesh. Present study focused on the rapid identification of live Mycobacterium tuberculosis among treatment failure cases. Materials and Methods: Sputum samples from a total of 100 category-I and category-II treatment failure cases, assumed as multidrug resistant tuberculosis, were studied through fluorescein diacetate (FDA) staining under light emitting diode (LED) fluorescence microscope. Considering culture method as gold standard, we also compared the results of FDA staining with that of auramine O staining. Results: A total of 85% acid-fast bacilli were detected by FDA staining, 82% by auramine O staining and a total of 85% isolates were detected in Lowenstein-Jensen (LJ) culture. The sensitivity of FDA staining (96.47%) was estimated to be slightly higher than that of auramine O staining (91.76%). Moreover, 76.47% cases were detected as multidrug resistant tuberculosis (MDR-TB). Conclusion: Taken together, FDA staining method has been proposed to be appropriate for the rapid diagnosis of drug resistant tuberculosis.

Comparison of Ziehl Neelsen and Auramine O staining methods on direct and concentrated smears in clinical specimens.

Background: In developing countries like ours with a large number of tuberculosis (TB) cases and limited resources, the diagnosis of TB relies primarily on smear microscopy for Acid Fast Bacilli (AFB) but its sensitivity is limited in paucibacillary cases. Aim: To evaluate the increase in efficacy of smear microscopy when smears are prepared from clinical samples after concentration by Petroff’s method and stained by Auramine O (AO) fluorescent dye as against Ziehl Neelsen (ZN) staining of similar taking culture as the gold standard. Methods: Smears were prepared from 393 clinical samples both by direct and after Petroff’s concentration and examined by fluorescent microscopy and Ziehl Neelsen method .The concentrated material was also cultured on Lowenstein Jensen media and the results of the two microscopy methods were compared with the culture results taken as the gold standard. Results: Mycobacterial growth was detected in 137(35.77%) specimens, out of which three were non-tubercular mycobacteria. Using culture as the reference method, the sensitivity of direct staining was 55.55% for ZN and 71.85% for AO. Direct fluorescent microscopy detected 9.29% paucibacillary sputum samples that were missed on ZN staining. On concentration, the sensitivity increased by 6.67% for ZN and 11.11% for AO. The sensitivity of AFB smear microscopy increased by 27.41% and was statistically significant (p=<.001) when both methods were combined. The specificity was 99.19% for both ZN and AO. Conclusion: Fluorescent microscopy has higher sensitivity and comparable specificity which is further enhanced by concentration. Now with the advent of newer inexpensive Light Emitting Diode (LED) based fluorescent microscopes (FM), which are easier to use, fluorescent microscopy can be widely used even in peripheral laboratories where culture facilities are not available.

A Study on Cryptosporidium Infection in Patients with Chronic Severe Hepatitis B / 中国医师杂志

Objective To explore the relationship between chronic severe hepatitis and cryptosporidium infection to provide evidences for scientific prevention and treatment of chronic severe hepatitis. Methods Fecal samples of 218 patients with chronic severe hepatitis B (CSHB) and 140 children with diarrhea were collected, and were examined for cryptosporidium oocytes by using auramine-phenol staining method (AA-p) and modified acid-fast staining method (MAF), and for cryptosporidium DNA by PCR and restriction digestion analysis. The factors affecting cryptosporidium infection of patients with CSHB were preliminarily analyzed. Results The positive rates of cryptosporidium infection detected by AA-p, MAF and PCR in the patients with CSHB and children with diarrhea were 4.1%, 3.2%, 6.0% and 0.7%, 0.7%, 1.4%, respectively. The positive rate of cryptosporidium infection detected by PCR in patients with CSHB was higher than that in children with diarrhea (P

Comparison of Stain Methods with PCR and Culture for the Detection of Mycobacterium Tuberculosis in the Sputum / 대한임상병리학회지

BACKGROUND: Currently, many laboratories have selected several different methods for the detection of M. tuberculosis in the sputum. To select efficient method for clinical laboratories among the various methods, we compared the results of several methods. METHODS: Total 72 sputums were examined by the six combinations of stain methods. The samples were constructed as follows on the result of direct smear ZN stain; negatives (26), traces (3), 1+(9), 2+(12), 3+(12) and 4+(10). The true positives were determined after close evaluation of the clinical, radiological and other laboratory findings. RESULTS: The sensitivities and specificities of each methods were as follows; direct smear ZN stain were 83.6% and 100%, direct smear Auramine stain were 90.9% and 100%, centrifugation ZN stain were 94.6% and 100%, centrifugation Auramine stain were 98.2% and 94.1%, cytocentrifugation ZN stain were 96.4% and 100%, cytocentrifugation Auramine stain were 100% and 64.7%, nested PCR were 80% and 94.1% and culture were 67.3% and 100% respectively. CONCLUSIONS: Concentration method by centrifugation is suitable for routine laboratory if enough centrifugal force were engaged. Auramine stain is more suitable staining method than ZN stain in direct smear but not in concentrated smear because it has the potency of false positivity. The PCR assay is thought to be not only a fast, sensitive method but also a specific method for the direct detection of M. tuberculosis in the sputum. The culture method using Ogawa media is specific but not sensitive.