Sensitivity of HPV tests on stained vs. unstained cervical smears

OBJECTIVE: To examine the effect of Pspanicolaou staining of cervical smears on the sensitivity of molecular biologic HPV tests. STUDY DESIGN: Two sensitive HPV tests were used, HPV DNA sequence analysis after polymerase chain reaction (PCR) amplification and the Hybrid Capture II method (HC II) (Digene Diagnostics Inc., Silver Spring, Maryland, USA). Papanicolaou-stained and unstained smears taken simultaneously were examined from 265 women readmitted for examination due to an atypical squamous cells of undetermined significance diagnosis. RESULTS: After an HPV test with the PCR method on unstained slides, 66% of the women were HPV positive, whereas the same women were HPVpositive in 54% when Papanicolaou-stained slides were analyzed. However, this difference was not statistically significant (p > 0.1). With the HC II method, 55% of unstained smears were HPV positive whereas 29% were HPV positive, when Papanicolaou-stained slides were examined. This difference was significant (p < 0.001). The same strong differences in sensitivity were observed when both the PCR and HC II methods were studied on the same Papanicolaou stained glass slides, whereas on unstained slides no significant difference was found. CONCLUSION: The results demonstrate that Papanicolaou staining of a cervical smear significantly decreases the sensitivity of an HPV test performed with the HC II method, whereas the PCR method is less affected. With the Papanicolaou method, the hematoxylin bath is followed by HCl treatment, and strong acid treatment destroys DNA.     

Blinded evaluation of DNA extraction and genotyping of stained Cryptosporidium on glass slides

AIMS: A blinded trial was performed on Cryptosporidium genotyping using polymerase chain reaction (PCR)/restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein (COWP) gene between DNA extracted from oocyst suspensions as compared with DNA from fixed and stained faecal smears on glass microscope slides. METHODS AND RESULTS: Sixty-five faecal smears on slides were stained by one of three different methods comprising 50 positives and 15 negatives as determined by the observation of Cryptosporidium oocysts by microscopy. The expected result in terms of detection and the COWP genotype detected was achieved using DNA extracted from 94% of the slides tested. CONCLUSIONS: This study shows that DNA, which can be amplified by PCR, is present in stained smears on glass microscope slides. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be useful for molecular epidemiological studies on a range of gastrointestinal pathogens where samples are collected from locations remote from the testing laboratory.     

Sensitivity and specificity of polymerase chain reaction in Giemsa-stained slides for diagnosis of visceral leishmaniasis in children

The aim of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) in the detection of Leishmania DNA in archived Giemsa-stained bone marrow slides for diagnosis of visceral leishmaniasis (VL), and to compare PCR with conventional diagnostic techniques, like direct microscopy and parasite culture. Specimens of archived Giemsa-stained bone marrow slides from 91 patients with VL and from 79 controls with other diseases or conditions were studied. PCR showed the highest sensitivity (92.3%) and had good specificity (97.5%). Direct examination detected 79.1% and culture 59% of positive samples. In addition, PCR was able to detect VL in 16 of 19 patients (84.2%) with negative microscopy. PCR in Giemsa-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may be useful in the diagnosis of difficult cases. Slide smears are easily stored, do not require special storage conditions such as low temperatures, and can be easily mailed to centers where PCR is available, making it an excellent option for diagnosis in the field.     

Isolation of high-molecular-weight DNA from small samples of blood having nucleated erythrocytes,collected, transported,and stored at room temperature

Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (phenol-chloroform extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies.     

Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence.

The sequence of the spliced-leader gene repeat of a Brazilian Trypanosoma vivax stock from cattle showed high similarity to sequences of West African T. vivax in both intron and intergenic sequences. This is the first evidence based on DNA sequences of close-relatedness between Brazilian and West African T. vivax stocks. A T. vivax-specific diagnostic PCR assay based on spliced-leader gene intergenic sequences was able to amplify DNA from T. vivax stocks from South America (Brazil, Bolivia, and Colombia) and West Africa. Species-specificity of this method was confirmed by results obtained by testing 15 other trypanosomes, including other species and subspecies that can also infect cattle. The PCR assay developed presented high sensitivity, detecting the DNA content of only one parasite and also revealing T. vivax infection in asymptomatic animals without detectable parasitemia by microhematocrit or in Giemsa-stained blood smears. Use of crude preparations from field-blood samples collected on both filter paper and glass slides as DNA template suggested that this method could be useful for the diagnosis of T. vivax in large epidemiological studies.     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

Ag-NOR technique in fine needle aspiration cytology of salivary gland masses.

Because of their complexity, salivary gland lesions are often difficult to identify correctly with fine needle aspiration cytology. To see whether the Ag-NOR staining technique for nucleolar organizer regions would be useful in this respect, we studied a series of smears from benign and malignant salivary gland lesions. The smears, previously treated with Papanicolaou and May-Grünwald-Giemsa stain, were destained and restained with Ag-NOR silver. The correlation between the cytologic-histologic diagnosis and the number of Ag-NORs in benign (sialadenitis, pleomorphic adenoma, oncocytoma and Warthin's tumor) and malignant lesions (adenoid cystic carcinoma, adenocarcinoma, carcinoma ex pleomorphic adenoma and squamous carcinoma) was statistically significant (P = less than .05). The Ag-NOR technique appears useful in the diagnosis of salivary gland lesions. One great advantage is that previously stained slides can be reused for silver staining, thus providing an excellent guide to the diagnosis, especially in doubtful cases and when corresponding histologic specimens or extra unstained slides are unavailable.     

IMPROVED METHODS FOR THE ISOLATION OF CYANOBACTERIAL DNA FROM ENVIRONMENTAL SAMPLES1

DNA isolated from environmental samples often contains enzyme inhibitors disruptive to downstream molecular applications. Most of the existing methods of cyanobacterial DNA isolation do not effectively eliminate these inhibitors from sediment samples or cells collected from freshwater ecosystems. We describe improved methods based on the xanthogenate‐SDS nucleic acid isolation (XS) method of Tillett and Neilan (2000) . Our improved methods provided high‐quality cyanobacterial DNA that could be amplified in PCR and digested with a restriction enzyme. Results were superior to several commercial kits. The DNA yield was also similar to that obtained via the standard XS method. These methods should provide valuable new tools for the expanded application of molecular genetics to limnological and oceanographic research.     

Cytomorphologic results of preparation experiments for monolayer deposition of cervical material.

For automated prescreening methods by high resolution analysis serving as a detecting method in gynecologic mass screening programs a new monolayer deposition method of cervical material has been used. This method will be outlines briefly and the mode of evaluation as well as current cytomorphological findings will be presented. With regard to measurability of the slides prepared according to the new method a number of cytologic criteria were thought to be of particular importance. These criteria are delineated and compiled in a table. A form in which these criteria were listed was filled in by cytopathologists for each slide evaluated. When performing isolation and centrifugation procedures several new morphologic questions arose to the cytopathologist which can only partly be answered by now. If taking into account all criteria of evaluation it may be followed from the present experiences that slides of cervical material are much more suited for automated prescreening methods by high resolution analysis if prepared after isolation and centrifugation in macromolecular liquids than are conventional Papanicolaou smears or slides from suspensions with isolated cells that were not subjected to centrifugation procedures.     

DNA cytofluorometry in micro-dissected paraffin embedded breast tissue: description of a method

DNA microfluorometry on smears obtained from paraffin embedded tissue has been shown to be a distinctive possibility. In this paper a simple method for the detachment of cells from mammary ducts and ductules is described. Areas of interest were selected in conventional slides stained with Haematoxylin and Eosin. The corresponding paraffin embedded block was then dewaxed. The areas under study were retraced with a stereomicroscope and the cells within ducts and ductules were scraped out with a 0.4 mm diameter fine needle. Cells were isolated with mechanical and enzymatic procedures and stained with the Feulgen reaction. The DNA content of single cells was then measured using a microfluorometer.     

一种适于PCR扩增的小麦基因组DNA快速提取法

许多小麦分子生物学研究需要对大量的小麦样品进行PCR检测,因此,建立一种快速提取小麦基因组DNA的方法十分必要。根据国外报道的一种快速提取水稻和玉米基因组DNA的方法,我们对部分提取步骤进行变动后,在小麦上进行了尝试,长度为1．5kb的片段能得到稳定的扩增。该方法样品研磨在1.5ml的离心管内进行,后续操作不用酚、氯仿、CTAB、SDS和巯基乙醇,整个提取过程不需要使用通风橱,操作步骤简单,花费时间少,而且提取的小麦基因组DNA完整性好,量也较可观。一个DNA样品可供50～100次PCR反应使用,适用于小麦遗传多样性、分子标记辅助选择、转基因后代检测以及引物筛选、分子标记定位等多种研究。     

Infantile chlamydial conjunctivitis. A comparison of Papanicolaou, Giemsa and immunoperoxidase staining methods

The use of conjunctival smears to diagnose infantile Chlamydia trachomatis infection increased sixteen-fold in our hospital between the years 1979 and 1984. The present study was conducted to compare Papanicolaou and Giemsa staining methods with the avidin-biotin technique of immunostaining utilizing a highly specific antichlamydial monoclonal antibody. On retrospective review of 33 patients, chlamydial infection was diagnosed in 61% of the Papanicolaou-stained and 64% of the Giemsa-stained slides. After the Papanicolaou-stained slides were destained and immunostained with the antichlamydial antibody, round particles corresponding in size to elementary and reticulate bodies were readily seen in 79% of the cases. In comparison with the immunoperoxidase method, the sensitivity and specificity of Papanicolaou staining were 73% and 86%, respectively, while the corresponding figures for Giemsa staining were 77% and 100%, respectively. The study established the applicability of the immunoperoxidase method to this clinical condition, confirmed the accuracy of diagnoses with routine stains and highlighted the increasing incidence of chlamydial conjunctivitis in our hospital population.     

Role of unstained smears in determining sample adequacy

OBJECTIVE: To determine adequacy with air-dried, unstained smears. STUDY DESIGN: The study was conducted on a total of 70 cases. The cases were divided into 2 groups. Group I consisted of all 70 and was analyzed by a consultant, observer A. Group II consisted of 41 cases from group I. In addition to observer A, a junior resident with 3 months' experience in pathology (observer B), analyzed the slides independently. The results were compared with those on stained smears. RESULTS: When correlated with stained smears, in group I, 55 of 58 (94.8%) cases were reported as adequate, and 11 of 12 cases (91.7%) were labeled inadequate. All were proven correct. In group II, stained smears confirmed that 33 of 35 (94.3%) were labeled adequate by observer A and 33 of 36 (91.7%) by observer B. Stained smears did not confirm 1 of 6 (16.6%) cases labeled inadequate by observer A and 1 of 5 (20%) cases by observer B. CONCLUSION: Unstained smear examination is an effective technique for determining adequacy. An inexperienced practitioner can perform it as well.     

The diagnosis of malaria and identification of plasmodium species by polymerase chain reaction in Turkey

More than half of the world's population is exposed to malaria in approximately 100 countries. Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria endemic areas. We have developed a PCR method to determine the presence of plasmodium DNA in blood. The method can also identify the species of the plasmodium by restriction enzyme analysis of the amplified product. We evaluated the performance of this method in the diagnosis of malaria suspected cases in Turkey by comparing to microscopy of the blood smears: blood samples were obtained from 114 patients with malaria symptoms, including fever and/or chills lasting for several days, before starting treatment. Thin and thick blood smears were prepared immediately in the region of specimen collection. After isolation of DNA from blood samples, DNA was amplified by PCR and digested by restriction enzyme AluI. The obtained fragments were analyzed by agarose gel electrophoresis. The number of parasites in the thick and thin smears of the blood samples was evaluated microscopically after staining by Giemsa and results were compared by PCR results. Among 114 plasmodium positive cases detected by microscopy, 100 were also detected by PCR. There were 14 false negatives and no false positive by PCR. Compared to microscopy, the sensitivity, specificity and Positive Predictive Value (PPV) of PCR were determined as 76%, 100% and 100%, respectively.     

Retrospective study of Chlamydia trachomatis using the polymerase chain reaction on archival Papanicolaou-stained cytologic smears.

OBJECTIVE: To detect chlamydial DNA on archived Papanicolaou-stained (Pap) smears using the polymerase chain reaction (PCR) technique. STUDY DESIGN: A PCR assay was designed to identify chlamydial DNA using consensus sequences unique to the genus Chlamydia in the 16S rRNA gene. This assay produced a 109 base pair product containing a single Pvu II restriction site. One hundred cervicovaginal Pap smears from a teen clinic population were processed for DNA isolation and PCR. Amplifiable DNA was isolated from 93 of the 100 cases as determined by a human growth hormone gene. These specimens were subjected to chlamydial PCR. RESULTS: PCR analysis of the 93 samples yielded 6 that were positive for the chlamydial 16S rRNA sequence. The six positive chlamydial amplicons were purified and subjected to Pvu II restriction enzyme analysis to validate their identity. The analysis confirmed the identity of the products, as a single Pvu II restriction site resulted in 41 base pair and 68 base pair products, as predicted. CONCLUSION: PCR testing for Chlamydia trachomatis can be performed on DNA isolated from archival Pap smears. Using this methodology, 6.5% of young women in our teen clinic population were positive for chlamydial DNA.     

一种高效提取普通小球藻叶绿体DNA的新方法

本文采用尿素-月桂酰肌氨酸钠（urea-sarkosyl）法, 用于分离带有坚硬细胞壁小球藻的高纯度叶绿体DNA （cpDNA）。将对数生长期的小球藻收集后置于冰上研磨, percoll密度梯度离心收集叶绿体层, 显微观察表明叶绿体经梯度离心后形态完整。采用尿素-月桂酰肌氨酸钠法、蛋白酶K消化及酚/氯仿/异戊醇抽提, 获得了高纯度的cpDNA。检测结果显示, cpDNA分子长度为22 kb, A260：A280值为1.87±0.01, 产率达（2.52±0.01） μg？g-1 （DW）; cpDNA编码的16S rDNA扩增呈阳性, 而由细胞核编码的18S rDNA扩增呈阴性。表明cpDNA纯度高, 没有受到核基因组DNA的污染, 符合小球藻cpDNA高通量测序的要求。同时, 该方法也适合提取具有相似细胞壁成分的其他微藻的基因组DNA和cpDNA。     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.     

Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus)

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.     

Detection and isolation of glycoproteins and nuclear proteins in the bovine brain

Using ammonium sulphate precipitation, ion exchange chromatography and preparative isoelectrofocusing, 9 organ-specific glycoproteins and 16 specific nuclear proteins were isolated from bovine brain nervous tissue in a homogeneous state. The isolation of proteins was controlled by a solid phase immunoenzymatic analysis. The molecular weight, subunit composition and isoelectric points of the proteins were determined and their ability to interact with immobilized calf thymus DNA and concanavalin A was demonstrated. It was assumed that the multiplicity of specific proteins of brain tissue is a molecular basis which provides for the functional specificity of the nervous tissue at large.