Comparison of Schistosoma mansoni irradiated cercariae and Sm23 DNA vaccines.

Immunization with defined antigens is generally less effective at inducing host protection against experimental infection with Schistosoma mansoni than vaccination with attenuated infective cercariae. We predicted that quantitative and/or qualitative differences existed between the immune responses generated to attenuated cercariae and those induced by defined antigens. Thus, we compared immune responses typically associated with protection in the murine model between animals vaccinated with attenuated cercariae and mice immunized with DNA encoding Sm23, a schistosome integral membrane protein that has previously been shown to confer protection. Mice vaccinated three times with attenuated cercariae demonstrated higher levels of protection than Sm23-vaccinated animals but spleen cells from Sm23 DNA vaccinated mice produced significantly higher levels of schistosome antigen-specific IFN-gamma. Both vaccines induced similar levels of Sm23-specific antibody and post-challenge dermal inflammation. However, the pulmonary inflammatory responses following challenge were much less pronounced in DNA immunized animals compared to those receiving irradiated cercariae. Thus, although Sm23 DNA vaccination effectively induced parasite-specific IFN-gamma and antibody responses, it failed to evoke other critical responses needed for optimal vaccine efficacy.

Evaluation of synthetic, M type-specific peptides as antigens in a multivalent group A streptococcal vaccine.

The recent development of emm gene sequence-based typing methodology has allowed group A streptococci (GAS) M serotype prevalence data to be determined. This information has been used to identify the components of a multivalent M protein peptide vaccine that could theoretically prevent most of the GAS-mediated diseases in the USA. In this study, we have evaluated in mice the immunogenicity and protective ability of multiple synthetic, M type-specific peptides, derived from the N-termini of three prevalent GAS serotypes (three peptides per serotype, total of nine peptides). At least one peptide, representing each of the three M types tested, was immunogenic. Five of the nine synthetic peptides tested, elicited an immune response in mice, and sera raised against four of the peptides, all possessed functional activity as demonstrated in a bactericidal assay. In vivo nasopharyngeal challenge experiments were carried out with peptides from the M1 (peptide M1-3) and M3 (peptide M3-2) proteins induced in vivo immune protection by reducing intranasal carriage. Reduction in colonization for M1-3 and M3-2 was 90% (P=0.02) and 66% (P<0.17), respectively. A reduction in colonization of 67% (P=0.03) was observed for M3-2 immunized mice when M43, a heterologous serotype, was used as the challenge strain. These results show the utility of synthetic, M type-specific peptides as antigens in a multivalent GAS vaccine.

Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States.

From October 4 to November 2, 2001, the first 10 confirmed cases of inhalational anthrax caused by intentional release of Bacillus anthracis were identified in the United States. Epidemiologic investigation indicated that the outbreak, in the District of Columbia, Florida, New Jersey, and New York, resulted from intentional delivery of B. anthracis spores through mailed letters or packages. We describe the clinical presentation and course of these cases of bioterrorism-related inhalational anthrax. The median age of patients was 56 years (range 43 to 73 years), 70% were male, and except for one, all were known or believed to have processed, handled, or received letters containing B. anthracis spores. The median incubation period from the time of exposure to onset of symptoms, when known (n=6), was 4 days (range 4 to 6 days). Symptoms at initial presentation included fever or chills (n=10), sweats (n=7), fatigue or malaise (n=10), minimal or nonproductive cough (n=9), dyspnea (n=8), and nausea or vomiting (n=9). The median white blood cell count was 9.8 X 10(3)/mm(3) (range 7.5 to 13.3), often with increased neutrophils and band forms. Nine patients had elevated serum transaminase levels, and six were hypoxic. All 10 patients had abnormal chest X-rays; abnormalities included infiltrates (n=7), pleural effusion (n=8), and mediastinal widening (seven patients). Computed tomography of the chest was performed on eight patients, and mediastinal lymphadenopathy was present in seven. With multidrug antibiotic regimens and supportive care, survival of patients (60%) was markedly higher (<15%) than previously reported.

Mouse model for Chagas disease: immunohistochemical distribution of different stages of Trypanosoma cruzi in tissues throughout infection.

Different stages of Trypanosoma cruzi are seen during mammalian infection. Histologic sections of infected hearts have shown amastigotes and, when using immunohistochemistry (IHC), parasite antigens; however, demonstration of trypomastigotes in these tissues has proven elusive. Using a mouse strain that develops chagasic cardiomyopathy (histologically similar to human infection) 70 days after injecting T. cruzi-Brazil strain, we studied the distribution of parasite stages and the extent of inflammation. All organs had varying amounts of mononuclear inflammation by day 10, which peaked between day 20 and day 30, and decreased by day 50. Amastigotes were detected in myocytes, histiocytes, acinar pancreatic cells, astrocytes and ependymal cells by day 10, and the number of amastigotes peaked on day 30. Immunohistochemistry demonstrated trypomastigotes in sinusoids, vessels and interstitial tissues of several organs between day 15 and 50. Abundant parasite antigens (granular staining) were detected in connective tissues throughout the infection. The burden of amastigotes and trypomastigotes during the acute phase seems to correlate with the degree of inflammation and granular staining in the chronic stage.

Leptospirosis mimicking acute cholecystitis among athletes participating in a triathlon.

Leptospirosis, a disease acquired by exposure to contaminated water, is characterized by fever accompanied by various symptoms, including abdominal pain. An acute febrile illness occurred in athletes who participated in an Illinois triathlon in which the swimming event took place in a freshwater lake. Of 876 athletes, 120 sought medical care and 22 were hospitalized. Two of the athletes had their gallbladders removed because of abdominal pain and clinical suspicion of acute cholecystitis. We applied an immunohistochemical test for leptospirosis to these gallbladders and demonstrated bacterial antigens staining (granular and filamentous patterns) around blood vessels of the serosa and muscle layer. Rare intact bacteria were seen in 1 case. These results show that leptospirosis can mimic the clinical symptoms of acute cholecystitis. If a cholecystectomy is performed in febrile patients with suspicious environmental or animal exposure, pathologic studies for leptospirosis on formalin-fixed, paraffin-embedded tissues may be of great value.

Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada).

Chlamydia pneumoniae has been associated with atherosclerosis and several other chronic diseases, but reports from different laboratories are highly variable and "gold standards" are lacking, which has led to calls for more standardized approaches to diagnostic testing. Using leading researchers in the field, we reviewed the available approaches to serological testing, culture, DNA amplification, and tissue diagnostics to make specific recommendations. With regard to serological testing, only use of microimmunofluorescence is recommended, standardized definitions for "acute infection" and "past exposure" are proposed, and the use of single immunoglobulin (Ig) G titers for determining acute infection and IgA for determining chronic infection are discouraged. Confirmation of a positive culture result requires propagation of the isolate or confirmation by use of polymerase chain reaction (PCR). Four of 18 PCR assays described in published reports met the proposed validation criteria. More consistent use of control antibodies and tissues and improvement in skill at identifying staining artifacts are necessary to avoid false-positive results of immunohistochemical staining. These standards should be applied in future investigations and periodically modified as indicated.

Gastrointestinal basidiobolomycosis in Arizona: clinical and epidemiological characteristics and review of the literature.

Gastrointestinal basidiobolomycosis (GIB) is an unusual fungal infection that is rarely reported in the medical literature. From April 1994 through May 1999, 7 cases of GIB occurred in Arizona, 4 from December 1998 through May 1999. We reviewed the clinical characteristics of the patients and conducted a case-control study to generate hypotheses about potential risk factors. All patients had histopathologic signs characteristic of basidiobolomycosis. Five patients were male (median age, 52 years; range, 37--59 years) and had a history of diabetes mellitus (in 3 patients), peptic ulcer disease (in 2), or pica (in 1). All patients underwent partial or complete surgical resection of the infected portions of their gastrointestinal tracts, and all received itraconazole postoperatively for a median of 10 months (range, 3--19 months). Potential risk factors included prior ranitidine use and longer residence in Arizona. GIB is a newly emerging infection that causes substantial morbidity and diagnostic confusion. Further studies are needed to better define its risk factors and treatment.

Genotypic, clinical, and demographic characteristics of children infected with Helicobacter pylori.

Helicobacter pylori isolates vary between geographic regions. Certain H. pylori genotypes may be associated with disease outcome. Thirty-eight children underwent diagnostic upper endoscopy at four medical centers and were retrospectively analyzed to determine if H. pylori virulence genes were associated with endoscopic disease severity, histologic parameters, and host demographics. The H. pylori virulence genotype was analyzed by a reverse hybridization line probe assay and type-specific PCR. Endoscopic ulcers or erosions were found in 17 (45%) patients, with 13 (34%) of these patients having antral nodularity. Histological gastritis, of varying severity, was present in all children. Four patients harbored more than one H. pylori strain: one subject had both cagA(+) and cagA-negative strains, while three patients harbored either two different cagA-negative strains (two children) or two cagA(+) strains (one child). There were 28 (74%) cagA(+) isolates; 19 were associated with the vacA s1b genotype, 7 were associated with the vacA s1a genotype, 1 was associated with the vacA s1c genotype, and 1 was associated with the s2 genotype. Of 14 cagA-negative isolates, 6 were vacA s2 genotype, 4 were vacA s1b, 3 were vacA s1a, and 1 was vacA s1c. Nine of ten (90%) Hispanics had similar H. pylori strains (vacA s1b,m1), and all Asian-Canadian children were infected by strains with vacA s1c genotype. No correlation between H. pylori strain and endoscopic or histopathologic abnormalities was found. This study provides a baseline framework of North American children and their H. pylori strains, serving as a powerful epidemiological tool for prospective investigations to better understand the transmission and evolution of diverse disease outcomes.

Pathologic studies of fatal cases in outbreak of hand, foot, and mouth disease, Taiwan.

In 1998, an outbreak of enterovirus 71-associated hand, foot, and mouth disease occurred in Taiwan. Pathologic studies of two fatal cases with similar clinical features revealed two different causative agents, emphasizing the need for postmortem examinations and modern pathologic techniques in an outbreak investigation.

Gastric atrophy and extent of intestinal metaplasia in a cohort of Helicobacter pylori-infected patients.

Atrophy and intestinal metaplasia (IM) are preneoplastic gastric lesions associated with Helicobacter pylori infection. Atrophy and IM are usually found together; however, the association between increasing degrees of severity of both atrophy and IM has not been evaluated completely. Two pathologists graded atrophy and IM using the visual analog scale of the Sydney classification in gastric biopsies from 368 H pylori-infected patients. Extent of IM also included determining the number of specimens affected. We then correlated the degree of atrophy with the degree and number of specimens affected with IM by calculating relative risks (RR) and 95% confidence intervals (95% CI). The mean number of biopsies examined from each patient was 6.5. Atrophy and IM were found more frequently in the antrum (85% and 75% of biopsies, respectively). One hundred thirty-eight patients had a combination of atrophy and IM, 48 had IM only, and 89 had atrophy only. Fifty-three subjects had mild atrophy and IM (RR = 1.57; 95% CI 1.2-2.1), 69 had moderate atrophy and IM (RR = 1.86; 95% CI 1.9-2.4), and 16 had marked atrophy and IM (RR = 2.47; 95% CI 1.8-3.3). The median number of biopsy specimens with IM increased from 0 in subjects with no atrophy to 3 in subjects with severe atrophy. The degree of IM correlated with the degree of atrophy; the median degree was 0.6 in subjects with no atrophy and increased to 2.32 in those with severe atrophy. Our data suggest that higher degrees of IM in an individual specimen and increasing number of specimens with IM are associated with moderate or severe degrees of atrophy.

Screening markers for chronic atrophic gastritis in Chiapas, Mexico.

Intestinal-type gastric adenocarcinomas usually are preceded by chronic atrophic gastritis. Studies of gastric cancer prevention often rely on identification of this condition. In a clinical trial, we sought to determine the best serological screening method for chronic atrophic gastritis and compared our findings to the published literature. Test characteristics of potential screening tests (antibodies to Helicobacter pyloni or CagA, elevated gastrin, low pepsinogen, increased age) alone or in combination were examined among consecutive subjects enrolled in a study of H. pylori and preneoplastic gastric lesions in Chiapas, Mexico; 70% had chronic atrophic gastritis. English-language articles concerning screening for chronic atrophic gastritis were also reviewed. Sensitivity for chronic atrophic gastritis was highest for antibodies to H. pylori (92%) or CagA, or gastrin levels >25 ng/l (both 83%). Specificity, however, was low for these tests (18, 41, and 22%, respectively). Pepsinogen levels were highly specific but insensitive markers of chronic atrophic gastritis (for pepsinogen I <25 microg/l, sensitivity was 6% and specificity was 100%; for pepsinogen I:pepsinogen II ratio <2.5, sensitivity was 14% and specificity was 96%). Combinations of markers did not improve test characteristics. Screening test characteristics from the literature varied widely and did not consistently identify a good screening strategy. In this study, CagA antibodies alone had the best combination of test characteristics for chronic atrophic gastritis screening. However, no screening test was both highly sensitive and highly specific for chronic atrophic gastritis.

Maternal and congenital syphilis in Bolivia, 1996: prevalence and risk factors.

OBJECTIVES: The present study was carried out in seven maternity hospitals to determine the prevalence of maternal syphilis at the time of delivery and the associated risk factors, to conduct a pilot project of rapid syphilis testing in hospital laboratories, to assure the quality of syphilis testing, and to determine the rate of congenital syphilis in infants born to women with syphilis at the time of delivery--all of which would provide baseline data for a national prevention programme in Bolivia. METHODS: All women delivering either live-born or stillborn infants in the seven participating hospitals in and around La Paz, El Alto, and Cochabamba between June and November 1996 were eligible for enrolment in the study. FINDINGS: A total of 61 out of 1428 mothers (4.3%) of live-born infants and 11 out of 43 mothers (26%) of stillborn infants were found to have syphilis at delivery. Multivariate analysis showed that women with live-born infants who had less than secondary-level education, who did not watch television during the week before delivery (this was used as an indicator of socioeconomic status), who had a previous history of syphilis, or who had more than one partner during the pregnancy were at increased risk of syphilis. While 76% of the study population had received prenatal care, only 17% had syphilis testing carried out during the pregnancy; 91% of serum samples that were reactive to rapid plasma reagin (RPR) tests were also reactive to fluorescent treponemal antibody-absorption (FTA-ABS) testing. There was 96% agreement between the results from local hospital laboratories and national reference laboratories in their testing of RPR reactivity of serum samples. Congenital syphilis infection was confirmed by laboratory tests in 15% of 66 infants born to women with positive RPR and FTA-ABS testing. CONCLUSION: These results indicate that a congenital syphilis prevention programme in Bolivia could substantially reduce adverse infant outcomes due to this disease.

A cluster of fulminant myocarditis cases in children, Baltimore, Maryland, 1997.

The true incidence of myocarditis in children is difficult to estimate because many mild cases go undetected. This study describes an unusual cluster of myocarditis cases that occurred in young children living in the greater Baltimore area between May and October 1997. A search of multiple comprehensive databases and interviews with area pediatric cardiologists were conducted to identify unreported cases and determine the background rate of myocarditis in the area. Seven cases of myocarditis were found as well as two with a similar clinical picture and myocardial fibrosis on tissue examination. Six case patients with active myocarditis and one child with fibrosis died. The case children were predominantly black (eight of nine) and male (seven of nine), with no identifiable risk factors. The disease was characterized by a fulminant course with malignant arrhythmias. The greatest number of pediatric myocarditis deaths reported in 1 year prior to 1997 was three. Myocardial tissues were examined using immunohistochemistry, in situ hybridization, and polymerase chain reaction but no etiologic agent was identified. This outbreak is unusual because of both the number of cases and the fulminant course of the disease in this group of children.

An outbreak of plague including cases with probable pneumonic infection, Ecuador, 1998.

During February and March of 1998, 12 sudden deaths were reported among residents of a high-Andean community in Ecuador. All 12 fatalities were members of the same extended family and some had apparent exposure to sick guinea-pigs. Following an initial investigation by public health officials, an additional death was reported in a nearby community in April, also associated with exposure to sick guinea-pigs. Blood samples from humans, dogs, and a rodent were tested for antibody to the F1 antigen of Yersinia pestis by passive haemagglutination assay. Tissue from rodents was subjected to direct fluorescent antibody staining using fluorescein-labelled monoclonal antibody to Y. pestis F1 antigen. Formalin-fixed specimens from the 2 autopsies were evaluated using a 2-step alkaline phosphatase immunoassay with a monoclonal antibody to Y. pestis F1 antigen, and tissues that had not been embedded in paraffin were tested for the presence of DNA encoding the F1 structural antigen by polymerase chain reaction. Serological evaluation of close contacts of the fatalities revealed positive titres to F1 antigen of Y. pestis, the aetiological agent of plague, in 3 contacts from the first community and 1 from the second. Immunohistochemical staining of tissues collected from 2 of the fatalities provided evidence that both had pneumonic plague. Five of 14 dogs found in the communities were seropositive for plague antibody, providing evidence of a recent epizootic plague in the area.

Helicobacter pylori gastritis in children and adults: comparative histopathologic study.

The inflammatory response to Helicobacter pylori in adults has been well characterized. In children, studies of H. pylori gastritis have been based on small numbers of patients and have used loose definitions of the inflammatory components. Comparative study of the inflammatory response to H. pylori in children and adults can help our understanding of the natural history of this infection. Using the Updated Sydney Classification, we performed a retrospective, blinded pathology review of gastric biopsy specimens from 42 children and 40 adults with H. pylori infection, and we quantified each inflammatory cell separately (neutrophils, lymphocytes, plasma cells, and eosinophils). Differences in inflammatory cell components of children and adults were assessed by logistic regression analysis. More children had marked amounts of H. pylori (P = .05) and mild degree of neutrophils (P = .02), plasma cells (P = .005), and eosinophils (P = .0001) compared with adults. No differences existed when quantifying mononuclear cells or atrophy. Ulcers and intestinal metaplasia were present only in adults. We found that the numbers of inflammatory cells present in H. pylori-infected biopsy specimens is different in children and adults. We hypothesize that these differences demonstrate how H. pylori infection evolves from an acute childhood infection to an adult chronic disease.

Testing umbilical cords for funisitis due to Treponema pallidum infection, Bolivia.

To establish the frequency of necrotizing funisitis in congenital syphilis, we conducted a prospective descriptive study of maternal syphilis in Bolivia by testing 1,559 women at delivery with rapid plasma reagin (RPR). We examined umbilical cords of 66 infants whose mothers had positive RPR and fluorescent treponemal antibody absorption tests. Histologic abnormalities were detected in 28 (42%) umbilical cords (seven [11%] had necrotizing funisitis with spirochetes; three [4%] had marked funisitis without necrosis; and 18 [27%] had mild funisitis), and 38 [58%] were normal. Of 22 umbilical cords of infants from mothers without syphilis (controls), only two (9%) showed mild funisitis; the others were normal. Testing umbilical cords by using immunohistochemistry is a research tool that can establish the frequency of funisitis due to Treponema pallidum infection.

Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs.

Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans.

The role of pathology in an investigation of an outbreak of West Nile encephalitis in New York, 1999.

An outbreak of encephalitis occurred in New York City in late August 1999, the first caused by West Nile virus in North America. Histopathologic and immunopathologic examinations performed on human autopsy materials helped guide subsequent laboratory and epidemiologic investigations that led to identification of the etiologic agent.

Comparison of polyvinyl alcohol fixative with three less hazardous fixatives for detection and identification of intestinal parasites.

Polyvinyl alcohol (PVA) containing the fixative mercuric chloride is considered the "gold standard" for the fixation of ova and parasites in the preparation of permanently stained smears of stool specimens. However, mercuric chloride is potentially hazardous to laboratory personnel and presents disposal problems. We compared three new alternative, nontoxic fixatives with PVA, analyzing ease of sample preparation and quality of smears. Sixty-eight fresh stool specimens were divided into aliquots and placed in each of four different fixatives: PARASAFE (PS) (Scientific Devices Laboratory, Inc., Des Plaines, Ill.), ECOFIX (EC) (Meridian Diagnostics, Inc., Cincinnati, Ohio), Proto-Fix (PF) (Alpha-Tec Systems, Inc., Vancouver, Wash.), and low-viscosity PVA fixative (PVA) (Meridian). Specimens were processed and stained according to each manufacturer's directions. Parasites were found in 31 of 68 slide preparations with PVA, 31 with PF, 30 with EC, and 30 with PS. Blastocystis hominis and Iodamoeba bütschlii were preserved in a readily identifiable state by all methods of fixation. However, some parasites were more easily identified with some of the fixatives because of differences in parasite distortion. For example, Entamoeba histolytica (Entamoeba dispar) was detected in 13 stools fixed with PF, 7 with PVA, and 6 with EC but none with PS. Likewise, Chilomastix mesnili was identified in 13 specimens fixed with PF, 8 with EC, and 5 with PVA but only 1 with PS, while Entamoeba coli was seen much less frequently with PS than with the other three fixatives. A dirty background was observed in 41% of specimens prepared with PS, whereas background quality was acceptable with other fixatives. Sample preparation was most rapid with PS, although the EC method involved the fewest steps. In conclusion, PVA and PF produced the least parasite distortion, while PS proved unsatisfactory for the identification of E. histolytica, E. coli, and C. mesnili. Both PF and EC appear to be acceptable, environmentally safe substitutes for PVA.