Rapid screening of urine for significant bacteriuria by Gram stain,acridine orange stain,and the Autobac MTS system

Urine specimens submitted for microbiologic examination were screened for evidence of bacteriuria by three rapid methods: Gram staining, acridine orange staining, and the Autobac MTS system. The screening results were compared with those obtained by semiquantitative colony counts on agar plates. In this comparative study 1055 urine specimens were examined, of which 146 (13.8%) had colony counts of ≥1 × 10⁵/ml. All thre urine screening methods detected this level of bacteriuria at a sensitivity of 98% and a specificity of 55.2% (acridine orange), 66.0% (Gram stain), and 83.2% (Autobac), respectively. Of the 1055 urine specimens examined, 185 (17.5%) had colony counts of ≥1 × 10⁴/ml, at which level the sensitivity of the three methods was 93% and the specificity was 56.7% (acridine orange), 68.0% (Gram stain), and 86.0% (Autobac), respectively. For any level of sensitivity, the Autobac urine screen was shown to be more specific than either the Gram stain or the acridine orange method. The acridine orange stain was the least specific urine screen, especially at the upper limits of sensitivity.     

Diagnosis of Helicobacter pylori colonization of the gastric mucosa. A prospective comparative study of direct test methods and validation of a new urease test

A prospective study including 119 patients submitted for routine endoscopy of the upper gastrointestinal tract was initiated to compare three commercial biopsy urease tests with regard to sensitivity and specificity in detecting Helicobacter pylori colonization of the gastric mucosa and their reaction velocity. Specific culture, microscopy after staining with methylene-blue, histologic search after modified Giemsa staining and the combined results of culture and histology ("true standard") served as reference methods. The sensitivity and specificity of all three tests were high: Angass urease test 92.0%/97.7%, Telen-Quick test 94.7%/100%, CLO-test 94.7%/100% (analysis of one antrum and one body biopsy in a single test kit). Telen-Quick and CLO-tests reacted faster than the Angass urease test, but a period of 24 hours was necessary for all three tests to detect "true negatives". Histology and microscopy were reliable reference methods concerning sensitivity and specificity, while culture was characterized by inferior sensitivity (78.6%) and high specificity (100%).     

Acridine orange leukocyte cytospin test for central venous catheter--related bloodstream infection: a pediatric experience

The aim of this study was to evaluate the acridine orange leukocyte cytospin (AOLC) test for the rapid diagnosis of septicemia caused by central venous catheters (CVCs), without removing the catheter, in a pediatric intensive care unit population. Twenty-six patients admitted in the pediatric intensive care unit of Azienda Ospedaliera "Ospedali Riuniti di Bergamo", Italy, were prospectively evaluated for CVC-related infection. Blood for culture was taken from all patients. Quantitative endoluminal cultures of the removed catheter tip by Cleri's technique and semiquantitative superficial cultures of the hub were performed. Gram staining and an AOLC smear were done according to Kite's technique. Four Staphylococcus CVC-related bloodstream infections were identified. CVC colonization was detected in 8 patients. Four had septicemia (Enterococcus faecalis, Escherichia coli, Klebsiella oxytoca, Candida glabrata) without CVC involvement. However, Gram staining and the AOLC test were negative in all cases. We conclude that cytocentrifugation and acridine orange staining of blood withdrawn by Kite's method from an in situ catheter, although simple, quick, and inexpensive, did not aid diagnosis in this pediatric population.     

Early detection of Leishmania promastigotes in dog bone marrow cultures by acridine orange stain

An acridine orange staining technique was evaluated in comparison with other well-known methods for the laboratory diagnosis of leishmaniasis. A higher number of promastigotes was found in Novy-MacNeal-Nicolle (NNN) cultures inoculated with canine bone marrow, when culture samples were stained with acridine orange vital stain, compared with those detected using either Giemsa staining or unstained wet mount examination. Based on our data the acridine orange stain is a useful and timely technique in reflecting the true numbers of microorganisms present in a culture and also enhances the visualization of the parasites. The present results warrant further studies with human samples from suspected leishmaniasis patients.     

Diagnosis of local infection of a burn by semiquantitative culture of the eschar surface.

The purpose of this study was to determine whether semiquantitative surface cultures of the burn eschar are as reliable or useful as the classic invasive biopsy culture method. We used eschars from patients with burns in an in vitro system. Lyophilized pigskin was used to validate our methodology. Because of its simplicity and high degree of sensitivity and specificity as compared with quantitative biopsy culture, semiquantitative surface culture has a place in burn wound surveillance.     

Comparative evaluation of zinc sulfadiazine and silver sulfadiazine in burn wound infection

One percent silver sulfadiazine has been commonly used as a topical antimicrobial agent after a burn injury. Incidence of burn wound colonization by Staphylococcus aureus in patients treated with silver sulfadiazine has spurred research for other agents. A topical preparation that contains zinc and sulfadiazine (Zad-G) was evaluated for in vitro antibacterial spectrum and in vivo efficacy. Muscle biopsy specimens of rats treated with Zad-G appear to have fewer colonies of S. aureus than groups treated with silver sulfadiazine. Topical therapy with Zad-G for patients with burns was comfortable, reduced wound infection, and was comparable to therapy with silver sulfadiazine. A topical Zad-G preparation that contains zinc sulfadiazine appears to be an effective alternative to silver sulfadiazine in the treatment of burn wounds.     

The use of a differential fluorescent staining method to detect bacteriuria

BACKGROUND: This report describes a differential staining method which distinguishes gram-positive from gram-negative bacteria in fluorescence. Gram-positive bacteria appear yellow and gram-negative bacteria appear green. MATERIAL AND METHODS: The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein, which together form a red/ green system. In this report we compared the accuracy of the differential fluorescent staining method and the Gram stain in screening for bacteriuria, as detected by conventional cultures. RESULTS: A total of 1487 urine samples were tested. 289 cultures were positive. 237 specimens grew a single organism at 10(5) and 10(4) CFU/ml. 224 smears were detected by the differential fluorescent staining method and 162 were detected by Gram stain. 1198 samples failed to grow organisms at 10(5) and 10(4) CFU/ml. 107 smears were falsely positive by the fluorescent staining procedure and 289 were falsely positive by the Gram stain. On the basis of the culture results, the sensitivity of the differential fluorescent staining method was 94.5% and that of the Gram stain 68.3%. The specificity of the fluorescent staining procedure was 91.6% and that of the Gram stain 75.8%. The positive predictive value and the negative predictive value of the fluorescent staining method were 67.6% and 98.8%, respectively. Those of the Gram stain were 35.9% and 92.3%, respectively. CONCLUSIONS: A wide range of microbiological and chemical techniques are available to identify bacteria in urine. This fluorescent staining method represents a simple, rapid, reliable method with low-running costs. The main advantage of this technique is that it enables the microbiologist to exclude the presence of bacteria in the urine within a short time after specimen receipt and to eliminate a large number of specimens for culture with significant cost saving. Another advantage of the method is that it allows to distinguish gram-positive from gram-negative bacteria in positive slides on the same day the sample is obtained. The stained smears were easily interpreted, even when the bacterial counts in the specimen were low.     

Comparison of surface swab cultures and quantitative tissue biopsy cultures to predict sepsis in burn patients: a prospective study

This study aimed at evaluating the possibility of predicting septicemia in burn patients by using wound surface and tissue culture techniques as well as blood cultures. Fifty patients with full-thickness burn wounds covering at least 10% of the total body surface area were included. Signs of septicemia were noted in 21 patients (42%) and 29 patients died (58%). The bacterial colonization of the burn wounds consisted mainly of Staphylococcus aureus and Pseudomonas aeruginosa. Sepsis was better correlated to quantitative burn tissue biopsy cultures than surface swab cultures but the time needed for processing limits its predictive and therapeutic value.     

A method for assaying biofilm capacity on polyurethane-coated slides.

Isolates of coagulase-negative Staphylococci (CNS) were examined for their ability to form biofilms on polyurethane-coated slides. These slides provided a smooth plastic coating simulating polymeric plastic surfaces of medical grade catheter tubing. Slides were placed into plastic conical tubes containing tryptic soy broth inoculated with 10(6) bacteria per mL. The tubes were then incubated at 37 degrees for 48 hours. After incubation, 1 of the slides was stained with a fluorescent acridine orange stain and the other with a safranin stain. The incubation tubes were also stained with safranin. Forty-eight percent of the 65 CNS isolates were found to form a biofilm using acridine orange staining. Forty percent of the 65 CNS isolates were found to form a biofilm using the safranin stain on slides, whereas only 34% were found to adhere on sides of plastic tubes. Increased sensitivity of the fluorescent stain was probably due to enhanced visualization of smaller numbers of bacteria on the plastic. This method using fluorescent stained plastic-coated slides was easier to visualize and interpret than the tube method.     

五种结核病实验室检测方法对结核病诊断价值的效果评价

目的评价涂片法、BACTECMGIT960快速培养法、改良罗氏培养法、荧光定量PCR法和斑点免疫层析法在结核病实验室快速诊断方法中的作用和地位。方法对1260例结核病患者（结核病组）和100例非结核病患者（非结核病组）的各类标本采用涂片法、快速培养法、改良罗氏培养法、荧光定量PCR法进行检测,同时分离患者外周血血清进行结核抗体检测,并对这五种检测方法的结果进行分析比较。结果五种实验室检测方法对结核病组阳性标本和非结核病组阴性标本的检测结果差异均有统计学意义（χ2=466．31,χ2=216．14,P均〈0．05）。对于结核病组的涂阳和涂阴标本,快速培养法和荧光定量PCR法检测的灵敏度及准确度均高于其他三种方法,而涂片法、改良罗氏培养法、快速培养法的特异性均为100．0％,高于荧光定量PCR法和斑点免疫层析法;快速培养法的阳性检出率均较改良罗氏培养法高,差异均有统计学意义（χ2=3387．00,χ2=4233．00,P均〈0．05）,平均培养时间也较改良罗氏培养法短;但快速培养法与荧光定量PCR法的阳性检出率差异均无统计学意义（P均〉0．05）。结论BACTECMGIT960快速培养法和荧光定量PCR法是诊断结核病快速、有效的检测方法,其阳性检出率高,且能缩短培养时间,BACTECMGIT960快速检测系统还能进行快速药物敏感性试验,为结核病的快速诊断提供依据。     

FQ-PCR检测肠黏膜结核杆菌DNA对肠结核的诊断价值

目的建立一种实时定量检测结核分枝杆菌（MTB）插入序列IS6110 DNA的方法,探讨其在肠结核（ITB）诊断中的价值。方法采用FQ-PCR技术对36例内镜活检ITB标本（30例石蜡、6例新鲜组织）、36例Crohn’s disease标本（16例石蜡手术标本,15例石蜡内镜活检标本,5例新鲜内镜活检组织）及34例阴性对照标本进行IS6110 DNA的实时定量检测,并比较上述标本抗酸染色结果。结果13例ITB抗酸染色阳性,CD无1例阳性,抗酸染色对ITB诊断的敏感性36.11%。MTBIS6110DNA检测结果：23例ITB（63.89%）阳性,6例CD（16.67%）阳性,另有1例阳性为升结肠腺癌癌旁正常组织。MTBIS6110 DNA在ITB与CD中的阳性率差异有统计学意义（P〈0.05）。FQ-PCR对ITB诊断的敏感性为63.89%,特异性83.33%。FQ-PCR敏感性显著高于抗酸染色（P〈0.05）。MTBIS6110 DNA阳性的ITB标本其定量结果范围为2.44×10^-4-2.26×10^1拷贝/细胞。结论FQ-PCR检测MTBIS6110 DNA是一种快速有效的ITB诊断方法,其定量范围广,检测灵敏度高。对活检组织少、病理改变不典型、抗酸染色阴性组织的诊断和鉴别诊断尤有意义。     

Helicobacter pylori Infection in Endoscopic Biopsy Specimens of Gastric Antrum: Laboratory Diagnosis and Comparative Efficacy of Three Diagnostic Tests

Aims and objectives The present study was undertaken to compare the diagnostic yield of three available test procedures for detecting Helicobacter pylori (H. pylori) infection in endoscopic biopsies.Methods H. pylori infection was sought in 150 patients referred for upper gastrointestinal (GI) endoscopy. Multiple (about six) biopsy specimens were taken from pyloric antrum in each patient. Two biopsy specimens were subjected to one minute endoscopy room test - OMERT (a modified form of urease test), two were sent for histopathological analysis, where multiple sections were subjected to Giemsa staining and two were sent for microbiological evaluation after Gram's staining of heat fixed biopsy material.Results H. pylori positivity using histology, microbiology and OMERT was observed to be 33%, 30% and 27% respectively. However, overall 40% patients were infected when the results from three test procedures were combined, as H. pylori positivity was repeated more than once by these procedures separately. Histology was found to be superior to other two tests in our study, especially when multiple sections were examined, for the distribution of the organism was patchy. Amongst the infected, H. pylori was seen in only 30% of all 3-8 sections cut from a biopsy, whereas in 70% it was noted in a single section only.Conclusion The study revealed that histology has the highest detection rate and can be chosen as the "gold standard" amongst the three low cost test procedures available at present in our setup.     

Skin lipopolysaccharide-binding protein and IL-1beta production after thermal injury

In response to a burn injury, skin can have an inflammatory response characterized by the production of inflammatory cytokines, recruitment of immune cells, containment of invading organisms, and clearance of noxious substances from the wound. Lipopolysaccharide-binding protein (LBP) is a molecule that is capable of coordinating all 4 functions; we previously found evidence that suggested that LBP is produced within surgical wounds. Because of the central role of LBP in the response to bacterial infection, as well as in the high rate of infection after burn injuries, we sought to determine whether a thermal injury could affect wound LBP production and thereby affect host responses against bacterial infection. Rats were given either a burn or a sham burn and were killed 24, 48, and 72 hours after the injuries. Wound specimens were assayed for bacterial counts and for the presence of LBP, messenger (m)RNA, and interleukin (IL)-1beta mRNA. Wound LBP mRNA was significantly upregulated at 24 hours in the group with burn injuries (P < .05; burn vs sham burn); this was followed by decreases at 48 and 72 hours. Immunohistochemistry showed LBP protein in the epidermis of animals with burns. Bacterial counts increased in the group with burn injuries (P < .05; burn vs sham burn) and continued to rise for 72 hours. IL-1beta mRNA levels were elevated at all time points in the group with burn injuries (P < .05). These results suggest an inverse correlation between burn wound LBP expression and bacterial wound counts. This failure to maintain local LBP production after severe thermal injury despite localized inflammation shown by high IL-1beta levels may predispose local wounds to bacterial invasion.     

Comparison of silver sulfadiazine 1%, mupirocin 2%, and fusidic acid 2% for topical antibacterial effect in methicillin-resistant staphylococci-infected,full-skin thickness rat burn wounds

Silver sulfadiazine 1%, mupirocin 2%, and fusidic acid 2% were compared to assess the antibacterial effect of a once-daily application on experimental rat 15% full-skin thickness burn wounds seeded 24 hours earlier with a 10 standard strain of methicillin-resistant staphylococci. The quantitative counts of seeded organism in burn eschar and subjacent muscle were determined at postburn day 7, beside the cultures of blood and lung biopsies. All tested topical agents were equally effective against methicillin-resistant in reducing local burn wound bacterial count and preventing systemic infection.     

Differences in Pain Patterns for Infected and Noninfected Patients with Burn Injuries

The management of pain is a primary issue in burn care. Patients hospitalized for burn injuries experience severe pain on a daily basis, immediately after the injury and during the healing of the burn wound. Our clinical experience is that the intensity of pain is increased by wound infection. The purpose of this study was to investigate retrospectively whether patients experience increased pain intensity in conjunction with wound infection. A total of 165 patients with burn injuries were included, 60 of whom were diagnosed with infection. The results of this study showed a significant increase in pain intensity in association with infection. An increase in pain is one of the factors to be considered among the many assessments, tests, and treatments for patients with burn injuries.     

Histologic study of artificial skin used in the treatment of full-thickness thermal injury

Integra artificial skin is an effective means of treatment for full-thickness burns. In extensive burn injury the use of such skin substitutes may become the treatment of choice. The artificial skin consists of a dermal substitute of bovine collagen and chondroitin-6-sulfate and an epidermal layer of synthetic polysiloxane polymer (Silastic). Serial biopsy specimens were obtained from 131 patients during a period of 7 days to 2 years after application. In this histologic study, six sequential phases of repair were discerned. In addition, there were occasional unusual histologic features, eosinophilic infiltration, and/or macrophage-derived giant cell formation in the wound area; however, such findings did not clinically correlate with a negative response to Integra artificial skin. Good repair was obtained, with rare exceptions. An intact dermis was achieved as well as definitive closure of a complete epidermal layer with a minimum of scarring.     

A rapid simple in situ hybridization method for herpes simplex virus employing a synthetic biotin-labeled oligonucleotide probe: A comparison with immunohistochemical methods for HSV detection

We examined 28 paraffin-embedded tissue specimens with histologic evidence of herpes virus infection by in situ hybridization (ISH) utilizing manual capillary action technology (Micro Probe Staining System) and a 21 base synthetic multibiotinylated oligonucleotide probe from the HSV glycoprotein C region. The results were compared to a rapid simple immunohistochemical (IHC) protocol for detection of HSV proteins. HSV was detected by ISH and IHC in all but one specimen which was shown to be positive for varicella zoster virus by direct fluorescent antibody studies. Hybridization signal was confined to the nucleus in all cases. Staining was identified in cells with early as well as late cytopathic effect. IHC produced intense nuclear and/or cytoplasmic signal in infected cells and stained in areas of necrosis which were otherwise spared by ISH. HSV was detected by IHC and/or ISH in 3/5 specimens with histology suggestive of, but not diagnostic for, HSV infection. Both techniques were sensitive and specific for HSV, resulted in rapid detection of the pathogen in routinely processed tissues, and may be useful in cases where the histologic impression is equivocal for HSV infection. ISH for HSV may be preferred because it can identify early HSV infection, which in turn can be treated with antiviral agents. © 1994 Wiley-Liss, Inc.     

大鼠角膜碱烧伤后热休克蛋白70的表达(英文)

背景:角膜碱烧伤后损伤修复受许多因素的影响,热休克蛋白可促进变性、损伤蛋白质的迅速恢复或清除。目的:观察大鼠角膜碱烧伤后热休克蛋白70的表达及其与角膜损伤修复的关系。方法:检查大鼠眼无炎症及其他病变后,奥布卡因滴眼液点眼2次,棉签吸除结膜囊液体,将统一规格直径5mm的滤纸片浸泡于1mol/L NaOH溶液中10s,然后置于大鼠角膜中央30s制作大鼠角膜碱烧伤模型。分别于碱烧伤后6h,1,3,7,14,21d取材。结果与结论:RT-PCR、免疫组织化学染色、Western blot结果均显示热休克蛋白70mRNA和蛋白在角膜碱烧伤后1d即开始升高,7d时达高峰,14d后开始下降。苏木精-伊红染色及电镜观察显示角膜损伤在烧伤后6h即较明显,烧伤后7d逐渐恢复。提示大鼠角膜碱烧伤后热休克蛋白70的表达与碱烧伤后角膜损伤修复过程一致,参与了大鼠角膜碱烧伤后细胞的自我保护及修复过程。     

Helicobacter pylori and Helicobacter heilmannii in children. A Bulgarian study

The aim of the study was to evaluate the prevalence of Helicobacter pylori and Helicobacter heilmannii among 321 children. Gram staining, urease test and culture were performed. Of all patients, 52.6% were H. pylori positive and 0.3% were H. heilmannii positive. H. pylori infection was associated with chronic gastritis in 57.1%, with duodenal ulcer in 75% and with non-ulcer dyspepsia in 25.6%. This infection was more frequent in children aged 11-18 years than in younger patients. Rapid urease test, culture and direct Gram staining showed 42.3, 96.5 and 78.2% sensitivity and 93.2, 100 and 84.6% specificity, respectively. H. pylori was detected in 60.2% of fresh versus 52.8% of frozen specimens and in 64.8% in gastric biopsy versus 25% in gastric mucus specimens. H. pylori growth was detected after nine to 10 days in 6.2% and after 11 days in 1.2%. Culture exhibited the best accuracy of the three diagnostic methods. Frozen biopsy specimens gave reliable H. pylori detection unlike gastric mucus specimens. Eleven days of incubation for H. pylori is recommended. The study confirms an early acquisition of H. pylori infection in Bulgaria. The incidence of H. heilmannii infection in childhood is uncommon but clinically important.     

A rapid screening method for routine female genital tract swabs

Many hospital laboratories process large numbers of female genital tract swabs to confirm or eliminate the common causes of vaginal discharge. Employing media for culture and acridine orange fluorescent stain for microscopy provides a sensitive, economic and time saving method for routine screening of these types of specimen.