Histopathological diagnosis of Japanese spotted fever using formalin-fixed,paraffin-embedded skin biopsy specimens Usefulness of immunohistochemistry and real-time PCR analysis

Japanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology.     

Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR.

It is demonstrated that a diagnostic PCR for Borrelia burgdorferi can be inhibited in the presence of more than 500 ng of host (monkey skin) DNA. The inhibitor is the host DNA itself. An acceptable value for analytical sensitivity can be obtained by diluting the skin-B. burgdorferi proteinase K lysate to a level below the inhibitory concentration of the host DNA. Dilution of the lysate may obviate the need for further DNA purification.     

Detection of herpesvirus-like DNA by nested PCR on archival skin biopsy specimens of various forms of Kaposi sarcoma.

AIMS: To detect herpesvirus-like DNA sequences, defining a new herpesvirus, human herpesvirus 8 (HHV8), in paraffin wax embedded skin biopsy specimens of the various forms of Kaposi sarcoma. METHODS: DNA was extracted from archival skin biopsy specimens of Kaposi sarcoma, other mesenchymal skin tumours and various inflammatory skin lesions of HIV seropositive and negative patients. HHV8 DNA was detected by using a nested PCR assay. Human beta-globin DNA served as an internal control. RESULTS: Twenty two samples of Kaposi sarcoma were analysed, comprising 12 of the endemic type, nine HIV associated and one transplantation related. HHV8 DNA was detected by nested PCR in all forms of Kaposi sarcoma. By contrast, no HHV8 DNA was detected in five mesenchymal skin tumours or nine biopsy specimens of unspecific inflammatory skin lesions of HIV seropositive and negative patients. CONCLUSIONS: Detection of HHV8 DNA in paraffin wax embedded tissue can be used to confirm a diagnosis of Kaposi sarcoma.     

Real-time PCR assay using fine-needle aspirates and tissue biopsy specimens for rapid diagnosis of mycobacterial lymphadenitis in children

A real-time PCR assay was developed to diagnose and identify the causative agents of suspected mycobacterial lymphadenitis. Primers and probes for the real-time PCR were designed on the basis of the internal transcribed spacer sequence, enabling the recognition of the genus Mycobacterium and the species Mycobacterium avium and M. tuberculosis. The detection limit for the assay was established at 1,100 CFU/ml of pus, and the specificity tests showed no false-positive reaction with other mycobacterial species and other pathogens causing lymphadenitis. From 67 children with suspected mycobacterial lymphadenitis based on a positive mycobacterial skin test, 102 samples (58 fine-needle aspirates [FNA] and 44 tissue specimens) were obtained. The real-time PCR assay detected a mycobacterial infection in 48 patients (71.6%), whereas auramine staining and culturing were positive for 31 (46.3%) and 28 (41.8%) of the patients. The addition of the real-time PCR assay to conventional diagnostic tests resulted in the recognition of 13 more patients with mycobacterial disease. These results indicate that the real-time PCR is more sensitive than conventional staining and culturing techniques (P = 0.006). The M. avium-specific real-time PCR was positive for 38 patients, and the M. tuberculosis-specific real-time PCR was positive for 1 patient. Analysis of 27 patients from whom FNA and tissue biopsy specimens were collected revealed significantly more positive real-time PCR results for FNA than for tissue biopsy specimens (P = 0.003). Samples from an age-matched control group of 50 patients with PCR-proven cat scratch disease were all found to be negative by the real-time PCR. We conclude that this real-time PCR assay with a sensitivity of 72% for patients with lymphadenitis and a specificity of 100% for the detection of atypical mycobacteria can provide excellent support for clinical decision making in children with lymphadenitis.     

Uttrastructure of skin biopsy specimens in lysosomal storage diseases: Common sources of error in diagnosis

Common sources of error in the diagnosis of lysosomal storage diseases by ultrastructural examination of skin specimens have been identified in a series of biopsies from 72 patients. Four principal factors have emerged as leading pitfalls and sources of error in diagnosis. First, the skin biopsy technique itself may lead to alterations of normal skin ultrastructure. Second, artifacts may be produced during fixation and preparation of tissue for electron microscopy. Third, cellular organelles and structures normally present in human skin may be mistakenly interpreted as pathological. Fourth, the use of cultured skin fibroblasts for ultrastructural identification of storage material is often accompanied by artifacts induced in tissue culture and is not recommended. Recognition of these common problems may aid interpretation of the fine structure of skin abnormalities. Furthermore, when skin biopsy specimens are used as the primary source of diagnostic material, correlation of both skin ultrastructure and assay for specific lysosomal enzymes in cultured dermal fibroblasts will facilitate diagnostic accuracy.     

The diagnosis of Wegener's granulomatosis from transbronchial biopsy specimens

It is widely believed that thoracotomy is necessary to obtain biopsy specimens adequate for the histopathologic demonstration of pulmonary Wegener's granulomatosis (WG). We report five patients with WG who were diagnosed by transbronchial biopsy (TBB). In three cases, a diagnosis of WG was made by TBB alone. In the other two patients, subsequent open lung biopsies confirmed the TBB findings but did not add essential diagnostic information. Our experience suggests TBB may be appropriate as the initial diagnostic procedure in selected cases of suspected WG. This approach requires an understanding of the diverse histologic features of WG and the correlation of clinical and pathologic data.     

Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens

We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing countries.     

Evaluation of three techniques for differential diagnosis of prostatic needle biopsy specimens.

AIMS: To determine whether acidic mucin staining, lectin histochemistry using Wisteria floribunda agglutinin, and immunohistochemistry using the monoclonal antibody EAB 903 are of benefit in distinguishing between hyperplastic and carcinomatous prostatic glandular tissue in needle biopsy specimens. METHODS: Formalin fixed, paraffin wax embedded prostatic needle biopsy specimens of benign and malignant tissue were examined. Alcian blue-periodic acid Schiff staining was performed on 33 benign and 34 malignant cases. Wisteria floribunda agglutinin (WFA) binding sites were demonstrated by the avidin-biotin peroxidase (ABC) technique with and without neuraminidase pretreatment on 34 benign cases and 32 malignant cases. EAB903 anticytokeratin antibody binding sites were demonstrated using both an indirect immunoperoxidase (IIP) technique and an avidin-biotin peroxidase complex method on seven benign and 31 malignant cases. RESULTS: Acidic mucin staining was found in 17 of 34 malignant glands and was weakly positive in five of 33 benign glands. WFA positivity before neuraminidase pretreatment was present in 29 of 32 malignant glands and in 19 of 34 benign glands. After neuraminidase all benign and malignant cases showed positivity. EAB 903 positivity was seen in 11 of 31 malignant glands using the IIP technique and in two of 31 malignant glands using the ABC technique. In seven benign cases there was positivity in all glands using the IIP method with predominant basal cell staining in three and superficial cell staining in four. In benign cases using the ABC method two cases were negative. CONCLUSIONS: None of the three methods studied showed sufficient sensitivity and specificity to allow their recommendation for routine diagnostic use.     

Diagnosis of cutaneous tuberculosis in biopsy specimens by PCR and southern blotting.

AIMS: To evaluate the use of a gene amplification and hybridisation method for detecting mycobacterial nucleic acid as a possible diagnostic method for cutaneous tuberculosis infection. METHODS: Biopsy specimens from 20 patients with various skin conditions of possible tuberculous aetiology were studied. Six patients had ulcerative nodules, seven lupiform lesions, two non-necrotic granulomas, one scrofulous lichen, one impetigo, one erythematosus lesions, one warty lesions, and one suspected tuberculous lipoma. Biopsy specimens were stained using Ziehl-Neelsen stain and cultured in Lowenstein-Jensen medium. DNA was extracted and then amplified by PCR using primers specific for the Mycobacterium tuberculosis complex. Specificity was confirmed by Southern blotting. RESULTS: Of the specimens, 30% were positive for mycobacteria on staining with Ziehl-Neelsen stain, 60% were culture positive and 85% PCR positive. Only 35.2% of specimens were positive with all three techniques. A further 32.5% were both culture and PCR positive. All PCR negative samples were also negative when cultured or stained with Ziehl-Neelsen stain. Of the PCR positive specimens, 29.4% were negative when cultured or stained. CONCLUSIONS: PCR, using suitable primers, is an efficient and sensitive method for the diagnosis of cutaneous tuberculosis.     

Histopathology and immunopathology of skin biopsy specimens in Mediterranean spotted fever.

Histology of skin lesions and demonstration in them of Rickettsia conorii by direct immunofluorescence test (DIF) are presented in 13 patients with Mediterranean spotted fever (MSF). The lymphohistiocytic vasculitis which dominated the picture is not specific, however, it could be suggestive for the diagnosis of rickettsiosis. By DIF we demonstrated rickettsial coccobacillary forms in all the patients: in 12 macular lesions and in one "tache noire". The diagnosis was also confirmed by indirect immunofluorescence test in each case. DIF test was shown to be sensitive, specific and reliable in early diagnosis of MSF.     

Rapid immunoperoxidase staining of axons for frozen section diagnosis of nerve biopsy specimens.

A new method of freezing and embedding a nerve biopsy specimen and staining it with the immunoperoxidase technique for neurofilaments was developed to overcome the difficulties normally encountered in the assessment of tiny portions of nerve. The method clearly shows the architecture of the nerve, the exact number and size of all axons present, and the degree of fibrosis present. The entire procedure may be accomplished in 20 minutes.     

Xpert MTB/RIF for rapid diagnosis of tuberculous lymphadenitis from fine-needle-aspiration biopsy specimens

This study demonstrates the excellent diagnostic accuracy of the Xpert MTB/RIF test in patients with tuberculous lymphadenitis. The test sensitivity and specificity were 96.7% (95% confidence interval [CI], 86.6 to 100%) and 88.9% (95% CI, 69.6 to 100%), respectively, and it correctly identified 6/6 (100%) of the cytology smear-negative/culture-positive cases and 1 of 2 (50%) rifampin-resistant cases.     

CT引导下经皮肺穿刺细胞学和组织学联合检查的诊断价值

目的探讨CT引导下经皮肺穿刺细胞学和组织病理学联合检查在肺部周围性和弥漫性病变诊断中的价值。方法回顾性分析370例CT引导下经皮肺穿刺标本,分析细胞学与组织病理学诊断方法的相关性,比较细胞学、组织病理学以及两者结合诊断的敏感性、假阴性率,分析细胞学分型的准确率。结果 370例肺穿刺标本中,组织病理学诊断为癌、恶性肿瘤、疑癌、异型和阴性的例数分别为177(47.84%)、22(5.95%)、16(4.32%)、12(3.24%)和143(38.65%),细胞学诊断相应的例数为166(44.87%)、10(2.70%)、16(4.32%)、49(13.24%)和129(34.87%),两种检测方法结果相关(P0.001)。细胞学诊断敏感性为80.00%(192/240),组织病理学诊断敏感性为89.58%(215/240),两者结合诊断敏感性为98.33%(236/240),细胞学诊断与组织病理学诊断的敏感性差异有显著性(P0.05),组织病理学与两者结合诊断敏感性差异有显著性(P0.05)。细胞学阳性病例分型准确率为66.15%(127/192)。术中及术后并发症为14.59%,其中气胸31例,针道出血或痰中带血23例,均经相应处理后好转。结论 CT引导下经皮肺穿刺对于肺部病变是安全的、高敏感性和高准确性的定性诊断方法,同时行细胞学和组织病理学检查可显著提高诊断率,临床应用价值高。     

Sensitivity of PCR targeting the IS2404 insertion sequence of Mycobacterium ulcerans in an Assay using punch biopsy specimens for diagnosis of Buruli ulcer

Punch biopsy specimens from Mycobacterium ulcerans disease lesions were used to compare the sensitivities and specificities of direct smear, culture, PCR, and histopathology in making a diagnosis of M. ulcerans disease in a field setting. PCR for the insertion element IS2404 was modified to include uracil-N-glycosylase and deoxyuridine triphosphate instead of deoxythymidine triphosphate to reduce the risk of cross contamination. The "gold standard" for confirmation of clinically diagnosed Buruli ulcer was a definite histological diagnosis, a positive culture for M. ulcerans, or a smear positive for acid-fast bacilli (AFB), together with a possible histological diagnosis. For 70 clinically diagnosed cases of M. ulcerans disease, the modified PCR was 98% sensitive and gave a rapid result. The sensitivities of microscopy, culture, and histology were 42%, 49%, and 82%, respectively. The use of a 4-mm punch biopsy specimen was preferred to a 6-mm punch biopsy specimen since the wound was less likely to bleed and to need stitching. Given adequate technical expertise and the use of controls, the PCR was viable in a teaching hospital setting in Ghana; and in routine practice, we would recommend the use of Ziehl-Neelsen staining of biopsy specimens to detect AFB, followed by PCR, in AFB-negative cases only, in order to minimize costs. Histology and culture remain important as quality control tests, particularly in studies of treatment efficacy.     

The histological diagnosis of chronic gastritis in fibreoptic gastroscope biopsy specimens

The advent of the fibreoptic gastroscope with biopsy facilities has provided the means of obtaining biopsy specimens under direct vision from any part of the stomach. This creates new opportunities for the study of chronic gastritis, and, in particular, its evolution, topographical location, and causal relationships. On the basis of an experience with more than 2,500 biopsy specimens we have outlined a method for their systematic examination and have proposed a classification of chronic gastritis. This classification includes the type of mucosa, the type and stage of activity of the gastritis, and the presence and type of metaplasia. The classification is sufficiently flexible to allow within it quantitative assessment of individual histological features.     

A simple microassay of prolyl hydroxylase applicable to skin punch biopsy specimens.

A method is described for the assay of prolyl hydroxylase. The method is rapid, avoids the use of vacuum distillation equipment and can be applied to small tissue samples such as skin biopsies.