Hybrid of sodium polytungstate polyoxometalate supported by the green substrate for photocatalytic degradation of auramine-O dye.

Nowadays, textile industries have severely polluted the ecosystem and water sources via disposal of highly thermo- and photo-stable dyes within the ecology that require practical strategies to remove them from nature. In studies, the photocatalytic disinfection technique has been shown to have widespread applications in indoor air, environmental health, detection, biological, biomedical, laboratory hospital, pharmaceutical food industry, plant safety, waste water, effluents disposal, and drinking water disinfection. Herein, the sodium polytungstate (SPT) polyoxometalate (POM) was synthesized through a multi-step production procedure and hence modified via employing a green protocol by using tartaric acid, glutamic acid, and kombucha solvent toward efficient and total complete removal of the highly toxic, stable, and carcinogenic auramine-O (AO) dye from aqueous media. In this regard, developed materials were well-characterized, and their photocatalysis performance for photodegradation of AO dye was examined. Achieved results showed that the optimum absorption conditions were achieved at pH of 5.0, 15 mg/L of AO concentration, 0.04 g of photocatalyst dosage, and 110 min irradiation time, where SPT and modified SPT via green protocol showed full desirability (desirability function (DF) index of 1) along with 71.75 and 100% removal percentage, respectively. Obtained results justified the superior photocatalytic role of the SPT POM and its derived nanocluster that can be used for the complete removal of highly stable dyes from aqueous media till reaching the drinking water standard.

Epichlorohydrin and tripolyphosphate-crosslinked chitosan-kaolin composite for Auramine O dye removal from aqueous solutions: Experimental study and DFT calculations.

Chitosan (Ch, a natural polymer) and kaolin (K, a natural mineral) composite (Ch-K) was produced with the help of two crosslinkers, epichlorohydrin and tripolyphosphate, and then moulded into uniform beads in tripolyphosphate solution. The synthesis was proved by the analyses involving FT-IR and SEM-EDX. The beads were then used as the natural adsorbent for removal of the auramine O (AO), a frequently-used industrial dye, in aqueous solutions. Adsorbent performance of the Ch-K composite for AO dye molecules was optimized: 500 mg L-1 at pH 7.5 at 25 °C. The Langmuir model found 0.118 mol kg-1 for the maximum adsorption capacity of the Ch-K and the D-R isotherm model showed that the nature of the adsorption process was physical. Kinetics of the adsorption could be explained by using both IPD (intraparticle diffusion) and PSO (pseudo second order) models. Thermodynamic parameters demonstrated that the behaviour of the adsorption was an endothermic and spontaneous. The activity of the composite adsorbent was recovered (88%) after the five sequential adsorption/desorption cycles. Supported by experimental findings, the results obtained from in silico modeling at M06-2X/6-31+G (d,p) level helped hypothesise a mechanism for the formation of the Ch-K composite, and shed some light onto the adsorption behaviour of AO dye by assuming several favourable intermolecular interactions.

Posterior Pharyngeal Wall Tuberculosis-A Forgotten Entity Mimicking Malignancy.

BACKGROUND: Tuberculosis (TB) of posterior pharyngeal wall (PPW), either primary or secondary, is extremely rare and can mimic malignancy in elderly patients. CASE REPORT: A 36-year-old female patient presented with complains of nonhealing ulcer at the back of her throat for 4 months. On examination, there was an ulceroproliferative growth covered with slough over PPW. Positron emission tomography scan showed uptake in PPW. There was no uptake in any other part of body. A biopsy from PPW lesion showed epithelioid cell granulomas with Langhans type giant cells. Auramine-rhodamine staining showed few beaded bacilli consistent with TB. CONCLUSION: Tuberculosis of PPW is an extremely uncommon entity and can mimic malignancy. It should be kept as one of the differential diagnosis as it can be cured solely by giving anti-tubercular therapy.

Label free detection of auramine O by G-quadruplex-based fluorescent turn-on strategy.

Auramine o (AO) is a synthetic dye used in paper and textile industries. Although it has been an unauthorized food additive in many countries due to its toxic and carcinogenic possibility, its illegal uses have been detected in certain food products such as pasta, semolina and spices and also in pharmaceuticals. The presence of AO in food products should be monitored, therefore, to minimize the negative health effects on consumers. In this study, a simple, highly sensitive and selective label free detection method was investigated for AO by G-quadruplex-based fluorescent turn-on strategy. The optimum fluorescent detection assay was achieved with a specific G-quadruplex DNA sequence, c-myc, at 400 nM in Tris-HCl buffer at pH 7.4. The linearity of fluorescence intensity depending on AO concentration ranged from 0 to 0.07 µM and LOD and LOQ were 3 nM and 10 nM, respectively. The G-quadruplex-based detection assay was highly specific for AO as compared to other two synthetic food colorings and successfully applied to determine AO in pasta, bulgur and curry powder with recoveries in the range from 70.33% to 106.49%. This G-quadruplex-based label free detection assay has a significant potential to be used in the detection of AO in food products.

Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O.

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59-) or mutant reticulocyte frequencies (RETCD59-) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.

Papanicolaou induced fluorescence, Ziehl-Neelsen and Auramine O stains on lymph node fine needle aspiration biopsy specimens from children: A comparative study.

BACKGROUND: The rapid diagnosis of extrapulmonary tuberculosis in children remains challenging. The presence of enlarged lymph nodes provides an opportunity to obtain diagnostic material through fine needle aspiration biopsy (FNAB). Mycobacterial culture, traditionally the reference standard, has a slow turnaround time and PCR-based methods are not widely available in developing countries. Direct visualization of mycobacteria on microscopy can be a rapid method to confirm the diagnosis. This study compared three staining methods to visualize mycobacteria. METHODS: Hundred FNAB specimens from persistently enlarged lymph nodes in children, clinically suspicious for tuberculosis, were evaluated for the presence of mycobacteria by three staining methods: Papanicolaou induced fluorescence (PIF) and Auramine O staining using fluorescence microscopy and Ziehl-Neelsen (ZN) staining using conventional light microscopy. These methods were evaluated against mycobacterial culture. RESULTS: PIF positivity was 30%, with 38% and 48% for Auramine O and ZN respectively. The combined ZN/PIF positivity was 56%. The highest diagnostic accuracy (73%) was demonstrated by ZN alone and in combination with PIF, with PIF alone showing the lowest (49%) accuracy. Although the combined test showed the highest sensitivity, it had the lowest specificity, while ZN was significantly more sensitive than both other staining modalities. No statistical difference in specificity was seen among the tests. CONCLUSION: This study suggests that Auramine O staining on previously ZN stained slides does not significantly improve diagnostic accuracy. While currently widely available methods of direct visualization of mycobacteria suffer from low sensitivity, the ZN stain remains a useful diagnostic test, particularly in resource-constrained countries.

Development and optimization of a novel sample preparation method cored on functionalized nanofibers mat-solid-phase extraction for the simultaneous efficient extraction of illegal anionic and cationic dyes in foods.

A simple and efficient three-step sample preparation method was developed and optimized for the simultaneous analysis of illegal anionic and cationic dyes (acid orange 7, metanil yellow, auramine-O, and chrysoidine) in food samples. A novel solid-phase extraction (SPE) procedure based on nanofibers mat (NFsM) was proposed after solvent extraction and freeze-salting out purification. The preferred SPE sorbent was selected from five functionalized NFsMs by orthogonal experimental design, and the optimization of SPE parameters was achieved through response surface methodology (RSM) based on the Box-Behnken design (BBD). Under the optimal conditions, the target analytes could be completely adsorbed by polypyrrole-functionalized polyacrylonitrile NFsM (PPy/PAN NFsM), and the eluent was directly analyzed by high-performance liquid chromatography-diode array detection (HPLC-DAD). The limits of detection (LODs) were between 0.002 and 0.01 mg kg-1, and satisfactory linearity with correlation coefficients (R > 0.99) for each dye in all samples was achieved. Compared with the Chinese standard method and the published methods, the proposed method was simplified greatly with much lower requirement of sorbent (5.0 mg) and organic solvent (2.8 mL) and higher sample preparation speed (10 min/sample), while higher recovery (83.6-116.5%) and precision (RSDs < 7.1%) were obtained. With this developed method, we have successfully detected illegal ionic dyes in three common representative foods: yellow croaker, soybean products, and chili seasonings. Graphical abstract Schematic representation of the process of the three-step sample preparation.

Auramine O, an incense smoke ingredient, promotes lung cancer malignancy.

Burning incense to worship deities is a popular religious ritual in large parts of Asia, and is a popular custom affecting more than 1.5 billion adherents. Due to incomplete combustion, burning incense has been well recognized to generate airborne hazards to human health. However, the correlation between burning incense and lung cancer in epidemiological studies remains controversy. Therefore, we speculated that some unknown materials in incense smoke are involved in the initiation or progression of lung cancer. Based on this hypothesis, we identified a major compound auramine O (AuO) from the water-soluble fraction of incense burned condensate using mass spectrometry. AuO is commonly used in incense manufacture as a colorant. Due to thermostable, AuO released from burned incenses becomes an unexpected air pollutant. AuO is classified as a Group 2B chemical by the International Agency of Research on Cancer (IARC), however, the damage of AuO to the respiratory system remains elusive. Our study revealed that AuO has no apparent effect on malignant transformation; but, it dramatically promotes lung cancer malignancy. AuO accumulates in the nucleus and induces the autophagy activity in lung tumor cells. AuO significantly enhances migration and invasive abilities and the in vitro and in vivo stemness features of lung tumor cells through activating the expression of aldehyde dehydrogenase family 1 member A1 (ALDH1A1), and ALDH1A1 knockdown attenuates AuO-induced autophagy activity and blocks AuO-induced lung tumor malignancy. In conclusion, we found that AuO, an ingredient of incense smoke, significantly increases the metastatic abilities and stemness characters of lung tumor cells through the activation of ALDH1A1, which is known to be associated with poor outcome and progression of lung cancer. For public health, reducing or avoiding the use of AuO in incense is recommended.

Application of ultrasonic radiation for simultaneous removal of auramine O and safranine O by copper sulfide nanoparticles: experimental design.

In this study, copper sulfide nanoparticles loaded on activated carbon (CuS-NP-AC) were synthesized by novel, low cost and green approach and characterized using SEM and XRD. The application of this material for the simulations removal of auramine O (AO) and safranine O (SO) from aqueous solutions was investigated. The dependency of removal percentages to variables such as pH, initial dyes concentration, adsorbent dosage, sonication time and sonication temperature were studied with response surface methodology (RSM) by considering the desirability function (DF). The quadratic model between the dependent and the independent variables was built. The proposed method showed good agreement between the experimental data and predictive value, and it has been successfully employed to removal of AO and SO in aqueous media. The studied adsorbent (0.06 g of CuS-NP-AC) was capable of high percentage removal (99.8% and 99.5%) of 18 mg mL(-1) AO and SO in short time (7.0 min).

Differences in the detection of Cryptosporidium and Isospora (Cystoisospora) oocysts according to the fecal concentration or staining method used in a clinical laboratory.

Despite the availability of many parasitological methods for detection of Cryptosporidium and Isospora (Cystoisospora) belli in fecal samples, there are uncertainties about the accuracy of these techniques in laboratory practice. In this study, 27 formalin-fixed positive stool samples for Cryptosporidium and 15 for I. belli were analyzed by 2 concentration methods, sedimentation by centrifugation (SC) and formalin-ethyl acetate (FE), and by 3 tintorial techniques, modified Ziehl-Neelsen (ZN), safranin (SF), and auramine (AR). No significant differences were observed on Cryptosporidium identification between concentration methods, while a significantly higher number of I. belli oocysts (P < 0.0001) was detected in fecal smears concentrated by the SC than by the FE method. Fecal samples processed by FE produced a median oocyst loss to the fatty ring of 34.8% for Cryptosporidium and 45.4% for I. belli. However, FE concentration provided 63% of Cryptosporidium and 100% of I. belli slides classified as superior for microscopic examination. Regarding the efficiency of staining methods, a more significant detection of Cryptosporidium oocysts was observed in fecal smears stained by ZN (P < 0.01) or AR (P < 0.05) than by the SF method. Regular to high-quality slides for microscopic examination were mostly observed in fecal smears stained with AR or ZN for Cryptosporidium and with SF or ZN for I. belli. This study suggests a great variability in oocyst power detection by routine parasitological methods, and that the most frequent intestinal coccidians in humans have specific requirements for concentration and staining.

Redox- and pH-responsive cysteamine-modified poly(aspartic acid) showing a reversible sol-gel transition.

Synthesis and characterization of a pH- and redox-sensitive hydrogel of poly(aspartic acid) are reported. Reversible gelation and dissolution are achieved both in dimethylformamide and in aqueous medium via a thiol-disulphide interconversion in the side chain of the polymers. Structural changes are confirmed by Raman microscopy and rheological measurements. Injectable aqueous solutions of thiolated poly(aspartic acid) can be converted into mechanically stable gels by oxidation, which can be useful for drug encapsulation and targeted delivery. Reduction-facilitated release of an entrapped drug from disulphide cross-linked hydrogels is studied.

[Nontuberculous mycobacterial infection in patients with cystic fibrosis: a multicenter prevalence study]. / Estudio multicéntrico de prevalencia de micobacterias ambientales en pacientes con fibrosis quística.

OBJECTIVE: To determine the prevalence of nontuberculous mycobacterial infection in patients with cystic fibrosis. PATIENTS AND METHODS: We performed a prospective study in which patients with cystic fibrosis were followed for 2 years; the patients were recruited from specialized units and were all over 6 years old. Sputum samples collected every 6 months were stained with auramine-rhodamine and cultures were prepared with a liquid and a solid medium. When stains or cultures were positive for nontuberculous mycobacteria, 1 or 2 additional sputum samples were obtained from the patients, who were monitored closely to assess the need for specific treatment. We assessed the following clinical variables: age, sex, presence of pancreatic insufficiency, use of aerosol antibiotic therapy, and long-term azithromycin and inhaled or oral corticosteroid therapies. RESULTS: A total of 220 patients (119 women) with a mean age of 22.62 years (range, 6-74 years) were enrolled; of these 23.6% were receiving azithromycin. We prepared 1303 sputum samples for mycobacterial growth (range per patient, 4-68 samples); 65 samples from a total of 17 patients (7.72%) were positive: 17 by auramine-rhodamine staining and 48 by culture. Eighty-eight culture samples were contaminated and Mycobacterium tuberculosis was not isolated in any of the cases. The mycobacteria isolated were M avium complex (n=10), M abscessus (n=6), and M fortuitum (n=1). Two or more positive cultures were obtained in 9 patients, 5 of whom experienced clinical deterioration and were prescribed specific treatment. No significant differences in clinical variables were found between patients with nontuberculous mycobacteria and those without. CONCLUSIONS: The prevalence of nontuberculous mycobacterial infection in patients with cystic fibrosis was not very high (7.72%), perhaps because azithromycin interfered with the growth of these bacteria. Patients with repeat isolations of mycobacteria should be monitored closely.

Métodos convencionais e moleculares para o diagnóstico da tuberculose pulmonar: um estudo comparativo / Conventional and molecular techniques in the diagnosis of pulmonary tuberculosis: a comparative study

OBJETIVO: Comparar quatro métodos laboratoriais no diagnóstico de tuberculose pulmonar. MÉTODOS: Foram realizadas pesquisa direta pelas colorações de Ziehl-Neelsen e auramina, cultura para micobactérias em meio Lõwenstein-Jensen (LJ) e polymerase chain reaction (PCR, reação em cadeia da polimerase) para Mycobacterium tuberculosis em 160 amostras de secreção respiratória de pacientes com suspeita de tuberculose pulmonar. As cepas isoladas foram identificadas por método radiométrico utilizando-se p-nitro-alfa-acetilamino-beta-hidroxipropiofenona (NAP) e métodos clássicos. A sensibilidade dos métodos foi comparada com o padrão ouro para o diagnóstico da tuberculose pulmonar, definido por critérios clínicos, radiológicos e microbiológicos. RESULTADOS: Dos 160 pacientes, 142 foram diagnosticados com tuberculose pulmonar de acordo com o padrão ouro. As técnicas de Ziehl-Neelsen e auramina, cultura em meio LJ e PCR apresentaram sensibilidade de 54,2 por cento, 58,4 por cento, 67,6 por cento e 77,5 por cento, respectivamente, quando comparados ao critério diagnóstico adotado. A especificidade dos quatro métodos foi de 100 por cento. A concordância na identificação da micobactéria entre PCR e o método radiométrico utilizando NAP foi alta (96,8 por cento). A sensibilidade da PCR foi de 50,8 por cento nas amostras com baciloscopia negativa e de 98,8 por cento naquelas com baciloscopia positiva. Nas amostras com resultados negativos na baciloscopia e cultura, a sensibilidade da PCR foi menor que nas com resultados positivos (25,6 por cento e 99,0 por cento, respectivamente). CONCLUSÕES: A PCR é método promissor no diagnóstico da tuberculose pulmonar, mesmo em amostras paucibacilares. Além disso, apresenta a vantagem da identificação simultânea e rapidez do resultado.

OBJECTIVE: To compare four laboratory methods in the diagnosis of pulmonary tuberculosis. METHODS: Respiratory secretion specimens were collected from 160 patients suspected of having pulmonary tuberculosis. Direct testing for Mycobacterium tuberculosis was carried out using Ziehl-Neelsen and auramine staining. In addition, culture in Lõwenstein-Jensen (LJ) medium and polymerase chain reaction (PCR) were used. The strains isolated were identified by means of a radiometric method using p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) and classical methods. The sensitivity of the methods was compared to the gold standard for the diagnosis of pulmonary tuberculosis, based on clinical, radiological and microbiological criteria. RESULTS: Of the 160 patients, 142 were diagnosed with pulmonary tuberculosis according to the gold standard. The sensitivity of Ziehl-Neelsen staining, auramine staining, culture in LJ medium and PCR was 54.2 percent, 58.4 percent, 67.6 percent and 77.5 percent, respectively, when compared with the diagnostic criterion adopted. All four methods presented 100 percent specificity. In the identification of mycobacteria, there was high (96.8 percent) concordance between PCR and the radiometric method using NAP. The sensitivity of PCR was 50.8 percent in samples with negative sputum smear microscopy results and 98.8 percent in those with positive results. The sensitivity of PCR was lower in specimens with negative results in sputum smear microscopy and culture than in those with positive results (25.6 percent and 99.0 percent, respectively). CONCLUSIONS: We found PCR to be a promising method for the diagnosis of pulmonary tuberculosis, even in paucibacillary specimens. Simultaneous identification and faster results are additional advantages of this method.

Cytological diagnosis of pulmonary nocardiosis in an immunocompromised patient.

We report a case of pulmonary nocardiosis in an immunosuppressed patient having vasculitis who presented with fever, cough and chest pain. A suspicion of nocardiosis was made on auramine O staining of material procured by CT guided fine needle aspiration cytology right lung. Modified Ziehl-Neelsen staining was useful in confirming the diagnosis. The patient showed remarkable recovery after treatment with co-trimoxazole. Quick identification of this uncommon pathogen in the cytological material using special stains helped in timely diagnosis and successful treatment of the patient.

Basis for treatment of tuberculosis among HIV-infected patients in Tanzania: the role of chest x-ray and sputum culture.

BACKGROUND: Active tuberculosis (TB) is common among HIV-infected persons living in tuberculosis endemic countries, and screening for tuberculosis (TB) is recommended routinely. We sought to determine the role of chest x-ray and sputum culture in the decision to treat for presumptive TB using active case finding in a large cohort of HIV-infected patients. METHODS: Ambulatory HIV-positive subjects with CD4 counts > or = 200/mm3 entering a Phase III TB vaccine study in Tanzania were screened for TB with a physical examination, standard interview, CD4 count, chest x-ray (CXR), blood culture for TB, and three sputum samples for acid fast bacillus (AFB) smear and culture. RESULTS: Among 1176 subjects 136 (12%) were treated for presumptive TB. These patients were more frequently male than those without treatment (34% vs. 25%, respectively; p = 0.049) and had lower median CD4 counts (319/microL vs. 425/microL, respectively; p < .0001). Among the 136 patients treated for TB, 38 (28%) had microbiologic confirmation, including 13 (10%) who had a normal CXR and no symptoms. There were 58 (43%) treated patients in whom the only positive finding was an abnormal CXR. Blood cultures were negative in all patients. CONCLUSION: Many ambulatory HIV-infected patients with CD4 counts > or = 200/mm3 are treated for presumptive TB. Our data suggest that optimal detection requires comprehensive evaluation, including CXR and sputum culture on both symptomatic and asymptomatic subjects.

Auramine phenol staining of smears for screening acid fast bacilli in clinical specimens.

Tuberculosis is a major threat to the public health allover world. Out of the total tuberculosis cases reported globally, more than half are reported from African continent and India. Two to three fold rises in tuberculosis cases has been reported in the last two decades. Early diagnosis and treatment is one of the effective tools to control the rapid spread of disease. The aim of this study was to find out the value of auramine phenol (AP) staining technique in diagnosis of the suspected tuberculosis cases. A total of 2000 samples which included sputum (746), gastric aspirates (380), urine (336), endometrial biopsy (150), pleural fulids (146), Synovial fluids (67), ascitic fluids (35), cerebrospinal fluids (43), bone marrow (18), lymph node biopsy (11), pericardial aspirates (6), skin biopsy (4), peritoneal fluids (2), and stool (1) were included in the study. Sample were subjected for decontamination procedure by using standard Petroffs method. The deposit smears were stained by auramine phenol (AP stain) and Ziehl-Neelsenstaining (ZN stain) and specimens were cultured for Mycobacterium tuberculosis. Of the total positive isolates 69.23% were having pulmonary tuberculosis and 30.76 had extrapulmonary tuberculosis Genitourinary tuberculosis was the most common diagnosis among the extrapulmonary tuberculosis followed by chronic synovitis, bursitis, meningitis, septic arthritis and pericardial effusion. Out of 130 positive samples 70 by culture, 66 smears were positive by auramine phenol stain and 62 were positive by ZN stain. A total of 27 samples were tested positive only by AP staining technique, which included (12) pulmonary and (15) extrpulmonary samples. The endometrial biopsy and pericardial fluid samples showed positive for acid fast bacilli by AP stain only, whereas ZN stain and culture technique failed to demonstrate any bacilli in the same sample. Auramine stain showed high sensitivity (47.14%) and specificity (96.58%). Result of the present study showed that the auramine stain is a better method for screening samples from the suspected cases of tuberculosis sample especially pulmonary and extrapulmonary cases where bacilli count is usually low.

Detection of Cryptosporidium oocysts by auramine and Ziehl Neelsen staining methods

Cryptosporidium spp is a common intestinal pathogen of animals and humans. It may have an important economic impact on farms and cause potentially zoonotic infections. Fecal specimens were collected from 331 domestic animals (81 beef cattle, 50 sheep, 100 pigs and 100 dogs) and checked for the presence of Cryptosporidium oocysts by way of Ziehl Neelsen and auramine staining methods. An overall positivity rate of 7.5 percent (25/331) was found, with rates of 10 percent (10/100) among the dogs and 18.5 percent (15/81) among the beef cattle. The feces of sheep and pigs tested negative. In beef cattle, 15 and 12 positive samples were detected by the auramine and Ziehl Neelsen staining techniques, respectively, with no statistically significant difference between the two methods. In dogs, the same number of positive samples was found by both techniques.

Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga.

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.

Comparison of the sodium hydroxide specimen processing method with the C18-carboxypropylbetaine specimen processing method using independent specimens with auramine smear, the MB/BacT liquid culture system, and the COBAS AMPLICOR MTB test.

A study was performed to diagnose tuberculosis by smear, culture, and nucleic acid amplification. The study was comprised of two independent arms. Each arm used a different specimen processing method; in one arm, all specimens were processed with N-acetyl-l-cysteine-sodium hydroxide, and in the other arm, all specimens were processed with C(18)-carboxypropylbetaine and lytic enzymes. In each arm, all processed sediments were split for analysis by auramine smear, by culture using the MB/BacT liquid culture system and solid media, and by nucleic acid amplification using the COBAS AMPLICOR MTB test. In the N-acetyl-l-cysteine-sodium hydroxide arm, 1,468 specimens were analyzed: 65 were smear positive; 88 and 42 were culture positive for Mycobacterium tuberculosis and nontuberculous mycobacteria, respectively; and 103 were PCR positive. Relative to cultures positive for M. tuberculosis, the sensitivity and specificity of the smear were 68.2% and 99.6%, respectively, and those of PCR were 75.0% and 97.3%, respectively. In the C(18)-carboxypropylbetaine study arm, 1,423 specimens were analyzed: 44 were smear positive; 82 and 31 were culture positive for M. tuberculosis and nontuberculous mycobacteria, respectively; and 91 were PCR positive. The sensitivity and specificity of the smear were 48.8% and 99.7%, respectively, and those of PCR were 78.0% and 98.0%, respectively. When the two arms were compared, C(18)-carboxypropylbetaine specimen processing significantly increased the number of smear-negative and culture-positive specimens and significantly increased the PCR sensitivity among this same group of specimens while at the same time significantly reducing the inhibition rate.

Auramine orange stain with fluorescence microscopy is a rapid and sensitive technique for the detection of cervical lymphadenitis due to mycobacterial infection using fine needle aspiration cytology: a case series.

OBJECTIVE: We sought to evaluate the effectiveness of the auramine orange (AO) stain in diagnosing mycobacterial cervical adenitis (MCA) from fine needle aspiration (FNA) cytology. METHODS: A retrospective review of 19 patients evaluated at 2 urban hospitals from 2000 to 2003 for suspected MCA. FNA specimens were inoculated to culture media and had direct smears stained by the auramine acid fast method. RESULTS: Mycobacteria were identified in 16 (84.2%) of 19 AO-stained FNA specimens, with results available within 4 hours. Corresponding cultures were positive for mycobacteria in 12 specimens, 9 tuberculous and 3 nontuberculous, and grew Mycobacterium tuberculosis from the 3 AO-negative specimens. Three of the 4 patients with negative cultures had previously taken anti-mycobacterial medications. CONCLUSION: The AO stain with fluorescence microscopy is a sensitive and rapid method for detecting tuberculous and nontuberculous mycobacteria. It is a valuable tool for the otolaryngologists and pathologists in the diagnosis of MCA.