Sucrose phosphate glutamate for combined transport of chlamydial and viral specimens

Sucrose phosphate glutamate (SPG) was evaluated as a transport medium for specimens submitted to the virology laboratory. The recovery rate in SPG and minimum essential medium (MEM) was compared at different temperatures and incubation times using herpes viruses. The results showed a successful recovery of viruses from clinical specimens and that SPG is equal to or better than MEM for maintaining herpes virus stability during transport.     

Comparison of processing techniques for detection of Pneumocystis carinii cysts in open-lung biopsy specimens.

Methenamine silver stain was used to compare the number of cysts of Pneumocystis carinii contained in lung concentrate smears of homogenized lung tissue with the number in impression smears. Results were also compared with histopathological examination of methenamine silver-stained paraffin-embedded sections. Of slides from 23 preparations, a greater number of cysts were contained in concentrate smears than in impressions (P less than 0.001). In four preparations, cysts were noted in concentrate smears only. All concentrate smears were positive, whereas 11 of the 23 histopathological sections were negative (P less than 0.01). The ability to detect the cyst phase of P. carinii in lung tissue is enhanced by the use of concentrate smears.     

Detection of Helicobacter pylori in gastric biopsy and resection specimens.

AIMS: To compare the sensitivity of detecting Helicobacter pylori in gastric biopsy and resection specimens using tinctorial and silver impregnation stains, immunohistochemistry and the polymerase chain reaction (PCR). METHODS: Formalin fixed, paraffin wax embedded tissue from 33 gastric biopsy specimens (26 showing chronic gastritis and seven showing low grade mucosa associated lymphoid tissue (MALT) lymphoma) together with blocks of uninvolved mucosa from gastrectomy specimens for MALT lymphoma (five cases) were studied. Consecutive sections were stained using haematoxylin and eosin, Giemsa, the Warthin-Starry silver stain, and a polyclonal antibody directed against H pylori using an immunoperoxidase technique following heat induced antigen retrieval. PCR analysis of DNA extracted from a further section was carried out using primers which amplified a 411 base pair fragment of the urease A gene. RESULTS: H pylori was detected in 14 (37%) sections stained with haematoxylin and eosin, 21 (55%) with Giemsa, 23 (61%) with Warthin-Starry, and 25 (66%) stained with the antibody. Seventeen (45%) cases were positive on PCR. Immunohistochemistry was positive in all cases in which H pylori was detected by other methods. CONCLUSION: Immunohistochemistry using an immunoperoxidase technique following heat induced antigen retrieval for detecting H pylori in gastric biopsy and resection specimens is highly sensitive and easy to use.     

Lymphocyte proliferative responses to chlamydial antigens in human chlamydial eye infections.

In order to study the relationship between cell-mediated immune responses to Chlamydia trachomatis and the pathogenesis of human chlamydial eye disease, we have measured the peripheral blood lymphocyte proliferative responses to whole chlamydial elementary bodies in 40 subjects with oculogenital chlamydial infection of varying severity, 13 subjects with genital chlamydial infections and 12 healthy seronegative controls. The mean stimulation index was significantly higher in those with oculogenital infections than in controls. There was a strong correlation between the response to C. trachomatis serotypes B and L1. We studied the relationship between proliferative responses and four clinical parameters: follicular conjunctivitis, papillary hypertrophy, corneal pannus and epithelial punctate keratitis, but were unable to show a significant association with any of these. Nor was there any association between proliferative response and serum antibody titre to C. trachomatis (pooled serotypes D-K), duration of disease or quantitative isolation of chlamydia from the conjunctiva. The depletion of CD8+ cells had no consistent effect on proliferative responses to serotype L1 in 13 subjects.     

Immune responses to chlamydial antigens in humans

Antibody titer, lymphocyte stimulation and leukocyte migration inhibition with chlamydial antigens were determined repeatedly over many months on human subjects. The volunteers were retrospectively placed into four groups on the basis of clinical, laboratory and epidemiologic criteria. Group A consisted of persons with proven or probable chlamydial infection, including an illness confirmed by chlamydial isolation or seroconversion, or a clinically compatible illness with positive serologic results. Group B were sexual partners or close contacts of group A individuals. Group C were laboratory workers with prolonged exposure to viable chlamydiae or their antigens. Group D included persons of comparable age as those in groups A and B, but lacking a history of symptomatic chlamydial infection or of contact with chlamydiae.Individual cases illustrated the rise of antibody and some cell mediated immunity reactions (CMI) with active chlamydial infection. By contrast, laboratory exposure resulted in elevation of CMI but not of antibody. Statistical analysis of the results in 46 volunteers tested repeatedly indicated a strong association of specific antibody with lymphocyte stimulation, but not with leukocyte migration inhibition. Regression analysis suggested that the type of exposure markedly influenced the relationship between antibody and lymphocyte stimulation. Measurement of immunotype-specific antibody titer by microimmunofluorescence (or an equally sensitive method) remains the best laboratory indicator of past chlamydial infection. Neither antibody nor CMI can, as yet, be definitely related to resistance to re-infection in humans.This work was supported by Public Health Service grant EY OO186 and NIH Hd 03939. We thank Hermine Keshishyan for her valuable technical assistance.     

Detection of endogenous type C viral antigens in mice during development

AKR and BALB/c mice (12- to 20- day embryos, newborn, and adult) were investigated by a radioimmunodiffusion (RID) method with test systems for group-specific antigen of the principal p30 structural protein (gs-1) of type C murine viruses and for type-specific antigen of Gross virus (AGLV). The p30 protein was clearly detectable after the 12th day of intrauterine development of both strains of mice; it persists in the tissues of the embryos until birth, and is found in the tissues of mice of both strains after the first day of postnatal development. AGLV was not detected in embryonic or adult BALB/c mice or in AKR embryos. However, this antigen was found in young AKR mice starting from the first to second day after birth. It is concluded from the results that the expression of p30 protein and AGLV is independent, within the limits of sensitivity of the RID method.Laboratory of Immunochemistry and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 7, pp. 72–75, July, 1977.     

Detection of chlamydial inclusions in cell culture or biopsy tissue by alkaline phosphatase-anti-alkaline phosphatase staining.

An immunological technique for detecting Chlamydia trachomatis and Chlamydia psittaci inclusions in infected McCoy cell cultures was developed by using a genus-specific monoclonal antibody to Chlamydia spp., rabbit anti-mouse immunoglobulin G bridging antibody, alkaline phosphatase-anti-alkaline phosphatase (APAAP) monoclonal antibody conjugate, and naphthol AS-phosphate/fast red substrate. Chlamydial inclusions stained red and were easily detected against a background of blue hematoxylin-stained nuclei. After 18 h, inclusions of C. trachomatis serovar L2 LGV434/Bu and C. psittaci strain 6BC were stained by APAAP but not by iodine or Giemsa. At 48 h inclusion counts were significantly higher in the APAAP cultures. Both the APAAP procedure and conventional staining detected 35 of 239 (15%) cultures 48 h after inoculation with urethral or endocervical specimens. However, at 24 h after inoculation 22 of 35 (63%) were positive by APAAP staining while negative by iodine. This immunostain also allowed identification of chlamydial inclusions in endometrial biopsies from patients with tubal factor infertility or pelvic inflammatory disease.     

Management and prevention of ocular viral and chlamydial infections

A majority of cases of preventable and/or curable ocular morbidity and blindness are caused by ocular infections. They may account for 70 to 90% of all ocular morbidity seen by family doctors, general practitioners, health centers, and local ophthalmologists in both developed and developing countries. Unfortunately, most health authorities and doctors, including ophthalmologists, consider these diseases to be of little or no importance because they are not fully aware of the high prevalence of these infections and the blinding sequelae which may occur following incorrect diagnosis and treatment. Also, they are not aware of the social and economic impact of these infections in the absence of proper management and implementation of preventive measures. In this review, we examine present knowledge of chlamydial and common viral ocular infections. We discuss the problems of diagnosis, management, and prevention and propose solutions relevant to developed and developing countries.     

Sponge artifact in biopsy specimens

We describe a sponge-induced artifact in histologic sections of small biopsy specimens. The artifacts are angulated, often triangular holes within the tissue. They appear to be introduced as individual sponge barbs become embedded in the perimeter of biopsy specimens during tissue processing. The artifact is generally of little importance, but in certain specimens, such as needle biopsies of the kidney or liver, it may occasionally obscure important information. Other methods, such as lens paper wrapping, may be superior in these situations. The utility of the tissue cassette sponge, in most situations, outweighs the artifact.     

Feasibility of histological grading and staging of chronic viral hepatitis using specimens obtained by thin-needle biopsy

We performed a retrospective study to investigate the feasibility of grading and staging of chronic viral hepatitis on specimens obtained by means of thin-needle biopsy (TNB; 20G) using the modified Ishak-system (J Hepatol 1995; 22:696-699). Specimens obtained using large-needle biopsy (LNB; 17G) served as a control. A total of 100 biopsy specimens from 88 patients were included in the study. Of the patients, 30 suffered from chronic hepatitis B, 54 from chronic hepatitis C, and 4 from both; 59 specimens were obtained by TNB and 41 by LNB. All four categories of the Ishak-system, i.e., interface hepatitis, confluent necrosis, lobular inflammation and portal inflammation, could be applied to TNB specimens and provided similar total scores to those observed in LNB specimens. Specimens obtained by TNB facilitated the diagnosis of liver cirrhosis. However, they bore the risk of underestimating the presence of cirrhosis in favor of advanced bridging fibrosis, whereas no differences were found in the overall recognition of liver fibrosis. Intra- and interobserver variabilities were not affected by the needle size. For the interobserver agreement, the kappa values for the category of inflammation ranged from 0.003 to 0.419 (TNB) and 0.096 to 0.470 (LNB) and for staging we noted kappa values of 0.351 (TNB) and 0.456 (LNB). Reproducibility increased when a tolerance of +/-1 was accepted for grading (total score) and staging; in this case, observer variability was less than 20%. This study showed that grading and staging of chronic viral hepatitis is feasible in TNB specimens and that intra- and interobserver variability poses a greater problem than needle size.     

Detection of viral antigens by solid phase radioimmunoassay on polyethylene film

Polyethylene film, without any pretreatment, may serve as a solid phase (SP) for RIA. Viral antigens (HBsAg, and influenza virus) are detected by SP-RIA on the film with a sensitivity of about 2-3 ng/ml or 40-60 pg/assay. The use of polyethylene film allows one to record RIA autographically. The use of micro amounts of reagents and specimens tested is an added advantage. No special equipment is necessary, the method is inexpensive, easy to perform and may be used for mass screening.     

Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens

Protein A-colloidal gold immunoelectron microscopy (PAG IEM) has been employed to specifically detect rotavirus and enterovirus antigen in negatively stained fluid specimens. Unlike other IEM methods, PAG IEM can detect not only viral antigen associated with morphologically recognizable particles but also viral antigens of unrecognizable ultrastructure. This rapid and sensitive immunoassay was found to be applicable to virus-infected stool specimens as well as partially purified virus preparations. The sensitivity of viral antigen detection by PAG IEM was 2- to 40-fold greater than direct IEM and 200- to 1,000-fold greater than direct electron microscopy. In addition, PAG IEM appears to offer a more reliable and sensitive alternative to standard IEM for detection and quantitation of viral antibody.     

Detection and identification of Mycobacterium tuberculosis in joint biopsy specimens by rpoB PCR cloning and sequencing

Osteoarticular tuberculosis (OAT) is an extrapulmonary tuberculosis and accounts for 1 to 3% of all tuberculosis cases. We used an rpoB PCR-plasmid TA cloning-sequencing method to detect and identify tubercle bacilli in surgical specimens from patients suspected of having OAT. By comparing the similarities of the rpoB sequences determined with those in GenBank, Mycobacterium tuberculosis was detected in 23 of 43 samples. Three of the 23 positive samples had mutations at codon 531, which are commonly observed in rifampin-resistant M. tuberculosis strains. Our results suggest that the rpoB PCR-TA cloning-sequencing method developed, which detects M. tuberculosis and which simultaneously determines its rifampin susceptibility, can also be used efficiently for the diagnosis of OAT.     

Detection of Ki-67 in liver biopsy specimens using monoclonal antibody to Mib-1

Ki-67 antigen was visualized in formalin-fixed, paraffin-embedded liver biopsy specimens using monoclonal antibody to Mib-1 to identify the proliferating hepatocytes. Thirty liver specimens obtained from 10 patients with chronic hepatitis (CH) or liver cirrhosis (LC) and 10 patients with hepatocellular carcinoma were studied. Liver specimens were treated with a pepsin solution or heated with autoclave or treated with microwave as a part of antigen retrieval system; then stained with an immunoperoxidase method using a monoclonal antibody to Ki-67 (Mib-1). Stable stainings were obtained in the sections treated with autoclave. Ki-67 was detected in the nuclei of hepatocytes, bile duct epithelium, fibroblast and infiltrating mononuclear cells. In patients with CH and LC, the numbers of hepatocytes positive for Ki-67 has a good co-relation with serum GPT level (p < 0.01), while has no relationship with the degree of fibrosis. The number of hepatocytes positive for Ki-67 has a good co-relation with the degree of the differentiation of hepatocellular carcinoma. Detection of proliferating hepatocytes using Mib-1 is useful to understand the degree of proliferation.     

Rapid cytologic diagnosis of cytomegalovirus interstitial pneumonia on touch imprints from open-lung biopsy

The diagnostic utility of Wright-Giemsa-stained touch imprints for identifying cytomegalovirus (CMV) in open-lung biopsies from marrow transplant recipients was studied. Of 41 consecutive biopsy specimens, 18 had culturally proven CMV, and 15 had typical intranuclear and intracytoplasmic inclusion bodies on permanent histologic sections. Wright-Giemsa-stained touch imprints from ten of the 15 biopsy specimens with CMV had characteristically enlarged cells containing 20-75 round to oval intracytoplasmic inclusions while only rarely containing intranuclear inclusions. The diagnosis of CMV from Wright-Giemsa-staining imprints (10/15) was superior to hematoxylin and eosin (H & E)-stained touch imprints (5/15) and H & E-stained frozen sections (2/15). Two cases of CMV had Papanicolaou-stained imprints that also demonstrated both intranuclear and intracytoplasmic inclusions. This simple technic of examining cytologic imprints from open-lung biopsy specimens will rapidly identify more than 50% of all confirmed cases of CMV, allowing earlier and possibly more successful antiviral therapy.     

Immunohistochemical detection of chlamydial antigens in association with cystitis.

To investigate the etiologic role of Chlamydia trachomatis in cystitis, the authors used the immunoperoxidase technique with a monoclonal antibody against Chlamydia and examined paraffin sections from 36 cases of histologically proven cystitis. The average patients' age was 60 (range, 2-85) years. Biopsies were taken for follow-up of treated bladder carcinoma (19), hematuria (8), and other nonneoplastic conditions (9). Chlamydial antigens were detected by immunohistochemistry in 12 (33%) of these 36 cases. Staining for Chlamydia occurred in the upper layers of the transitional epithelium and involved long stretches of epithelium. Underlying inflammation was usually chronic but did not have specific distinguishing features. Eight of the Chlamydia-positive biopsies were taken for follow-up of treated carcinoma, two were for hematuria, one for neurogenic bladder, and one for evaluation of sterile pyuria. Eleven (92%) of these 12 positive cases had a history of recent urologic instrumentation, in contrast to only 11 (46%) of 24 negative cases (P less than 0.02). There was no significant difference in the age or sex distribution between the two groups. The authors conclude that Chlamydia trachomatis can ascend the urethra and infect the bladder urothelium. Urologic instrumentation enhances the ability of Chlamydia to reach the bladder. Chlamydia trachomatis may play an etiologic role in cystitis.     

Detection of human papillomavirus in matched cervical smears and biopsy specimens by non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis. METHODS: HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear. RESULTS: An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration. CONCLUSIONS: These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.     

Transport of viral specimens.

The diagnosis of viral infections by culture relies on the collection of proper specimens, proper care to protect the virus in the specimens from environmental damage, and use of an adequate transport system to maintain virus activity. Collection of specimens with swabs that are toxic to either virus or cell culture should be avoided. A variety of transport media have been formulated, beginning with early bacteriological transport media. Certain swab-tube combinations have proven to be both effective and convenient. Of the liquid transport media, sucrose-based and broth-based media appear to be the most widely accepted and used. Studies on virus stability show that most viruses tested are sufficiently stable in transport media to withstand a transport time of 1 to 3 days. Some viruses may withstand longer transport times. In many cases, it is not necessary to store virus specimens in a refrigerator or send them to the laboratory on wet ice or frozen on dry ice. However, the specimen should not be exposed to environmental extremes. Modern viral transport media allow for more effective use of viral culture and culture enhancement techniques for the diagnosis of human viral infections.