The respiratory pathology in infants with sudden unexpected deaths in whom respiratory specimens were initially PCR-positive or PCR-negative for Bordetella pertussis.

BACKGROUND: In a previous controlled study, we investigated the relationship between Bordetella pertussis infections and sudden unexpected deaths among German infants (sudden infant death syndrome, SIDS). In this present study, we investigated further the respiratory pathology in a subset of infants in the original study. METHODS: Originally, there were 234 infants with SIDS and, of these, 12 had either a nasopharyngeal swab (NPS) or a tracheal swab specimen (TS) that was positive for B. pertussis by polymerase chain reaction (PCR). Here, tissue specimens from eight infants who were originally PCR-positive were compared with tissue specimens from seven infants in whom the original PCR studies were negative. RESULTS: The histopathologic diagnoses were as follows: 14 of 15 had pulmonary edema and the remaining case had early diffuse alveolar damage. Although 14 of 15 cases had some histologic or clinical evidence suggesting respiratory tract infection, the features were more consistent with a viral etiology, and in none were the findings typical of respiratory disease attributable to B. pertussis. CONCLUSIONS: The findings in this present investigation do not support a direct role of B. pertussis at the site of infection (ciliated epithelium) in the causation of SIDS. The clinical aspects of this study were carried out in the 1990s when pertussis was widespread in Germany. Therefore, the original finding of some PCR-positive cases is not surprising. The possibility that B. pertussis infection could still be a factor in some SIDS cases, e.g., by a systemic release of toxins, cannot be definitely ruled out.

Tissue diagnosis of Ehrlichia chaffeensis in patients with fatal ehrlichiosis by use of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

In the United States, human ehrlichiosis is a complex of emerging tick-borne diseases caused by 3 distinct Ehrlichia species: Ehrlichia chaffeensis, Ehrlichia ewingii, and the human granulocytotropic ehrlichiosis agent. Ehrlichioses are characterized by a mild to severe illness, and approximately 4% of cases are fatal. Because these obligate intracellular bacteria are difficult to resolve with routine histologic techniques, their distribution in tissues has not been well described. To facilitate the visualization and detection of ehrlichiae, immunohistochemistry (IHC), in situ hybridization (ISH), and polymerase chain reaction (PCR) assays were developed by use of tissues from 4 fatal cases of E. chaffeensis infection. Evidence of E. chaffeensis via IHC, ISH, and PCR was documented in all 4 cases. Abundant immunostaining and in situ nucleic acid hybridization were observed in spleen and lymph node from all 4 patients. Significantly, in 2 of these patients, serologic evidence of infection was absent. Use of IHC, ISH, and PCR to visualize and detect Ehrlichia in tissues can facilitate diagnosis of ehrlichial infections.

Transmission of Black Creek Canal virus between cotton rats.

Black Creek Canal (BCC) virus is a hantavirus associated with hantavirus pulmonary syndrome in southeastern North America. The virus was isolated from the spleen of a cotton rat (Sigmodon hispidus) trapped in southern Florida. Our previous studies have shown that we could consistently infect male cotton rats with BCC virus in the laboratory. These animals became persistently infected and virus could be detected in salivary glands, urine, and feces. In this report we show: (1) female and male cotton rats are equally susceptible to BCC virus infection, (2) susceptibility to infection was not influenced by age, (3) all inoculated rats transmitted the infection to uninoculated cage mates, and (4) offspring of infected rats became infected despite the presence of high maternal antibodies. The course of BCC virus infection, as determined by antibody response and the ability to isolate or detect virus, appeared to be similar regardless of whether the rats obtained their infection by inoculation or contact with inoculated rats. J. Med. Virol. 60:70-76, 2000. Published 2000 Wiley-Liss, Inc.

Hidden mortality attributable to Rocky Mountain spotted fever: immunohistochemical detection of fatal, serologically unconfirmed disease.

Rocky Mountain spotted fever (RMSF) is the most severe tickborne infection in the United States and is a nationally notifiable disease. Since 1981, the annual case-fatality ratio for RMSF has been determined from laboratory-confirmed cases reported to the Centers for Disease Control and Prevention (CDC). Herein, a description is given of patients with fatal, serologically unconfirmed RMSF for whom a diagnosis of RMSF was established by immunohistochemical (IHC) staining of tissues obtained at autopsy. During 1996-1997, acute-phase serum and tissue samples from patients with fatal disease compatible with RMSF were tested at the CDC. As determined by indirect immunofluorescence assay, no patient serum demonstrated IgG or IgM antibodies reactive with Rickettsia rickettsii at a diagnostic titer (i.e., >/=64); however, IHC staining confirmed diagnosis of RMSF in all patients. Polymerase chain reaction validated the IHC findings for 2 patients for whom appropriate samples were available for testing. These findings suggest that dependence on serologic assays and limited use of IHC staining for confirmation of fatal RMSF results in underestimates of mortality and of case-fatality ratios for this disease.

Congenital syphilis in a newborn: an immunopathologic study.

A 3-week-old girl presented to the emergency room with respiratory distress and generalized maculopapular rash. The newborn was hospitalized with a presumptive diagnosis of congenital syphilis, but she died after 2 days of therapy. Tissue from the gastrointestinal tract, brain, liver, spleen, and lung was studied by using direct fluorescent antibody and immunohistochemical analysis (IHC) for Treponema pallidum. The inflammatory infiltrate was characterized by using IHC against CD3, CD20, CD68, and smooth muscle actin. The diagnosis of congenital syphilis was confirmed by demonstrating spirochetes in tissues with IHC and direct fluorescent antibody examination. IHC showed abundant treponemes in the small intestine and liver and occasional spirochetes in the meninges. Bacteria were seen as intact spirochetes, granular staining, or large extracellular collections of antigen. A constant pathologic feature throughout the tissues was concentric macrophage (CD68-positive) infiltrate around vessels, giving an onion-skin appearance. IHC identified the macrophages as the prime immune response in congenital syphilis.

A novel immunohistochemical assay for the detection of Ebola virus in skin: implications for diagnosis, spread, and surveillance of Ebola hemorrhagic fever. Commission de Lutte contre les Epidémies à Kikwit.

Laboratory diagnosis of Ebola hemorrhagic fever (EHF) is currently performed by virus isolation and serology and can be done only in a few high-containment laboratories worldwide. In 1995, during the EHF outbreak in the Democratic Republic of Congo, the possibility of using immunohistochemistry (IHC) testing of formalin-fixed postmortem skin specimens was investigated as an alternative diagnostic method for EHF. Fourteen of 19 cases of suspected EHF met the surveillance definition for EHF and were positive by IHC. IHC, serologic, and virus isolation results were concordant for all EHF and non-EHF cases. IHC and electron microscopic examination showed that endothelial cells, mononuclear phagocytes, and hepatocytes are main targets of infection, and IHC showed an association of cellular damage with viral infection. The finding of abundant viral antigens and particles in the skin of EHF patients suggests an epidemiologic role for contact transmission. IHC testing of formalin-fixed skin specimens is a safe, sensitive, and specific method for laboratory diagnosis of EHF and should be useful for EHF surveillance and prevention.

Ebola (subtype Reston) virus among quarantined nonhuman primates recently imported from the Philippines to the United States.

In April 1996, laboratory testing of imported nonhuman primates (as mandated by quarantine regulations) identified 2 cynomolgus macaques (Macaca fascicularis) infected with Ebola (subtype Reston) virus in a US-registered quarantine facility. The animals were part of a shipment of 100 nonhuman primates recently imported from the Philippines. Two additional infected animals, who were thought to be in the incubation phase, were identified among the remaining 48 animals in the affected quarantine room. The other 50 macaques, who had been held in a separate isolation room, remained asymptomatic, and none of these animals seroconverted during an extended quarantine period. Due to the rigorous routine safety precautions, the facility personnel had no unprotected exposures and remained asymptomatic, and no one seroconverted. The mandatory quarantine and laboratory testing requirements, put in place after the original Reston outbreak in 1989-1990, were effective for detecting and containing Ebola virus infection in newly imported nonhuman primates and minimizing potential human transmission.

Immunohistochemical and in situ localization of Crimean-Congo hemorrhagic fever (CCHF) virus in human tissues and implications for CCHF pathogenesis.

BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal disease that occurs in parts of Africa, Asia, and eastern Europe, and that is caused by a recently emerged bunyavirus. Rapid laboratory diagnosis of CCHF infection is essential and is currently performed by virus isolation and serology. Histopathologic studies have been limited to a small number of cases, and little is known about the cellular tropism of CCHF virus and the pathogenesis of this disease. DESIGN: We conducted a retrospective case analysis of 12 patients with a diagnosis of CCHF infection, confirmed by virus isolation, who were evaluated at the Special Pathogens Unit, National Institute for Virology, South Africa. The clinicopathologic features of CCHF and the diagnostic role of virus isolation as compared with serology, immunohistochemistry, and in situ hybridization were evaluated. Additionally, the distribution of CCHF virus in human tissues was examined. RESULTS: The clinical and histopathologic features of CCHF resemble those of other viral hemorrhagic fevers. Of the 12 patients with virus isolation-confirmed CCHF infection, 5 were positive by serology, 10 by immunohistochemistry, and 5 by in situ hybridization. Immunohistochemistry and in situ hybridization analyses showed that the mononuclear phagocytes, endothelial cells, and hepatocytes are main targets of infection. Association of parenchymal necrosis in liver with viral infection suggests that cell damage may be mediated by a direct viral cytopathic effect. CONCLUSIONS: The diagnosis of CCHF, suspected by history and clinical features, can be supported histopathologically. However, since the pathologic features resemble those of other viral hemorrhagic fevers, an unequivocal diagnosis can be made only by laboratory tests. The utility of immunohistochemistry as a sensitive and rapid diagnostic modality was established by the high degree of concordance with virus isolation. Infection of mononuclear phagocytes, endothelial cells, and hepatocytes may play a critical role in the pathogenesis of CCHF.

Retrospective diagnosis of hantavirus pulmonary syndrome, 1978-1993: implications for emerging infectious diseases.

OBJECTIVE: To investigate the occurrence of unrecognized cases of hantavirus pulmonary syndrome preceding the detection of the 1993 outbreak in the southwestern United States and the initial description of the syndrome. DESIGN: Retrospective clinicopathologic and immunohistologic study. PATIENTS: Eighty-two patients who died prior to April 1993 with histologically unexplained noncardiogenic pulmonary edema. METHODS: Clinicopathologic review and immunohistochemical evaluation of autopsy tissues for evidence of hantaviral infection. RESULTS: Twelve retrospective fatal cases of hantavirus pulmonary syndrome were identified through clinicopathologic review and immunohistochemical testing of tissues. Patients' ages ranged from 16 to 49 years. The earliest identified case occurred in 1978, 15 years prior to the outbreak of hantavirus pulmonary syndrome in the southwestern United States. Immunohistochemical testing showed widespread deposition of hantaviral antigens, primarily within endothelial cells, similar to the pattern observed with current hantavirus pulmonary syndrome cases. CONCLUSIONS: Although hantavirus pulmonary syndrome was first recognized in 1993, the findings from this study document the earlier existence of this disease. These findings underscore the need for systematic archiving and analysis of clinical information and specimens from patients with diseases of unknown etiology to facilitate the study of new clinical entities and their associated etiologic agents.

Evaluation of idiopathic esophageal ulceration for human immunodeficiency virus.

Recent studies suggest human immunodeficiency virus (HIV) may be the cause of HIV-associated idiopathic esophageal ulcer (IEU). However, other causes of esophageal disease in HIV-infected patients have not been evaluated for appropriate comparison. Over a 14-month period 13 patients with IEU as determined by clinical, endoscopic, and pathologic criteria were identified. During the same period nine HIV-infected patients with cytomegalovirus (CMV) esophagitis and one HIV-infected patient each with herpes simplex virus esophagitis and gastroesophageal reflux disease (GERD) were also identified. Polymerase chain reaction (PCR) and in situ DNA hybridization (ISH) were performed on paraffin-embedded tissue formed on paraffin-embedded tissue of endoscopic biopsies of ulcer tissue using standard techniques. Eleven of 13 IEU patients (85%) as compared to seven of nine patients (78%) with CMV had HIV detected by PCR (P = 0.38). HIV was also detected in ulcer tissue from biopsy material from the patient with GERD but not herpes simplex virus esophagitis. In PCR-positive patients, ISH confirmed the presence of HIV in four patients (57%) with CMV and eight (73%) with IEU (p = 0.31). HIV was found only in inflammatory cells and not squamous epithelial cells. Given the similar prevalence of detection of HIV by PCR and ISH in ulcer tissue from both groups of HIV-infected patients as well as the location in rare inflammatory cells, we conclude that HIV infection of squamous mucosa does not appear to be the primary cause of IEU.

Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease.

A recent outbreak of a severe pulmonary disease in the southwestern United States was etiologically linked to a previously unrecognized hantavirus. The virus has been isolated from its major reservoir, the deer mouse, Peromyscus maniculatus, and recently named Sin Nombre virus. Clinically, the disease has become known as the hantavirus pulmonary syndrome (HPS). Since May 1993, 44 fatal cases of HPS have been identified through clinicopathological review and immunohistochemical (IHC) testing of tissues from 273 patients who died of an unexplained noncardiogenic pulmonary edema. In 158 cases for which suitable specimens were available, serological testing and/or reverse transcription-polymerase chain reaction (RT-PCR) amplification of extracted RNA was also performed. IHC, serological, and PCR results were concordant for virtually all HPS and non-HPS patients when more than one assay was performed. The prodromal illness of HPS is similar to that of many other viral diseases. Consistent hematological features include thrombocytopenia, hemoconcentration, neutrophilic leukocytosis with a left shift, and reactive lymphocytes. Pulmonary histopathological features were similar in most of the fatal HPS cases (40/44) and consisted of an interstitial pneumonitis with a variable mononuclear cell infiltrate, edema, and focal hyaline membranes. In four cases, however, pulmonary features were significantly different and included diffuse alveolar damage and variable degrees of severe air space disorganization. IHC analysis showed widespread presence of hantaviral antigens in endothelial cells of the microvasculature, particularly in the lung. Hantaviral antigens were also observed within follicular dendritic cells, macrophages, and lymphocytes. Hantaviral inclusions were observed in endothelial cells of lungs by thinsection electron microscopy, and their identity was verified by immunogold labeling. Virus-like particles were seen in pulmonary endothelial cells and macrophages. HPS is a newly recognized, often fatal disease, with a spectrum of microscopic morphological changes, which may be an important cause of severe and fatal illness presenting as adult respiratory distress syndrome.

Tumor necrosis factor-alpha induces circular forms of human immunodeficiency virus type-1 DNA in the persistently infected low-level expressing cell line, ACH-2.

The low human immunodeficiency virus type-1 (HIV-1) expressing T-cell line, ACH-2, was used to investigate accumulation of the circular, extrachromosomal form of HIV DNA (HD) after tumor necrosis factor-alpha (TNF-alpha) induction. We chose the 2 long terminal repeat (LTR) circular form to analyze unintegrated HD by polymerase chain reaction (PCR), using primer pairs which flank the 2 LTR HD. Approximately a 10-fold increase in 2 LTR HD was detected intracellularly in the TNF-alpha-induced ACH-2 cells using an end point-dilution assay. To examine the cellular compartment location of the 2 LTR HD accumulation, ACH-2 cells were fractionated into cytoplasmic and nuclear components and further subjected to PCR. A 4- to 5-fold increase in the 2 LTR HD signal was observed in the nuclear fraction. These results indicate that unintegrated HD increases in a chronically infected cell line after TNF-alpha induction. This phenomenon, which previously had been observed only with acute infections, may offer insight into basic pathogenic mechanisms.

Studies of myeloperoxidase gene expression at the cellular level by in situ hybridization.

Recent studies have demonstrated myeloperoxidase (MPO) gene expression during granulocytic differentiation. Since these studies have been done exclusively by Northern and dot blot analysis and frequently with mixed populations of cells, quantitative changes in gene expression for particular populations of cells are difficult to assess. We therefore examined MPO expression at the cellular level in various normal and malignant hematopoietic cells by the in situ hybridization (ISH) technique. Using this approach, we demonstrated that inducing the promyelocytic HL-60 cell line to differentiate along either monocytic or granulocytic pathways decreases MPO mRNA expression. Similarly, when ISH was performed on normal bone marrow, relatively high levels of MPO mRNA were detected in myeloblasts, promyelocytes, and early eosinophilic precursors, whereas the expression was markedly decreased in more advanced stages of myeloid differentiation. These findings agree with the known decrease in MPO protein synthesis observed during granulocytic differentiation and suggest that regulation of MPO protein synthesis occurs at the level of MPO mRNA expression. We conclude by showing that ISH can detect MPO mRNA in myeloblasts of patients with acute leukemia and can be a potentially useful technique in the study of myeloid differentiation in acute leukemias.

Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues.

We stained rabies-infected nervous and salivary-gland tissues fixed in formalin or acetone and embedded in paraffin with the avidin-biotin peroxidase system. With this system, rabies-virus antigen was detected in neurons, glandular acinar cells, and vascular endothelial cells more effectively than by immunofluorescence, especially when tissues were enzyme-digested with pronase before immunoperoxidase staining. The avidin-biotin peroxidase system should be useful for routine diagnosis, retrospective studies of rabies, and identification of specific cells involved in the spread of virus in rabies-infected hosts.

Immunofluorescent staining of Treponema in tissues fixed with formalin.

Immunofluorescent examination of formalin-fixed tissue for Treponema pallidum has generally been unsatisfactory because of nonspecific background fluorescence and poor contrast. We examined the process of treating deparaffinized formalin-fixed tissue sections with 1% ammonium hydroxide (NH4OH) to improve fluorescent staining. Treponema pallidum- and Treponema pertenue-infected rabbit testes or human tissue biopsy specimens fixed in 10% buffered formalin and embedded in paraffin were examined. Sections were cut one week to five years after embedment. Tissues were then stained with fluorescein- or rhodamine-labeled human anti- T pallidum globulin for 30 minutes at 37 degrees C. Treponemes were consistently stained and background staining was generally reduced after NH4OH treatment in both fresh and stored tissue. Cutting sections at a thickness of approximately 2 micron was critical to achieve optimal fluorescence.

Rapid demonstration of Legionella pneumophila in unembedded tissue. An adaptation of the Giménez stain.

The Giménez stain, originally developed for demonstrating rickettsiae, readily stained the Legionnaires' disease bacterium (Legionella pneumophila) in frozen tissue sections and smears of fresh or formalin-fixed lung tissue from patients who had confirmed Legionnaires' disease. With the Giménez procedure, the bacterium stained bright red against a blue-green background. The tissue Gram procedures also stained L. pneumophila in frozen sections and smears, but the staining reaction was weak, and these stains were neither as sensitive nor a consistent as the Giménez procedure.