Widespread outbreaks of clam- and oyster-associated gastroenteritis. Role of Norwalk virus

Consumption of raw shellfish has long been known to be associated with individual cases and sporadic outbreaks of enteric illness. However, during 1982, outbreaks of gastroenteritis associated with eating raw shellfish reached epidemic proportions in New York State. Between May 1 and December 31, there were 103 well-documented outbreaks in which 1017 persons became ill: 813 cases were related to eating clams, and 204 to eating oysters. The most common symptoms were diarrhea, nausea, abdominal cramps, and vomiting. Incubation periods were generally 24 to 48 hours long, and the duration of illness was 24 to 48 hours. Bacteriologic analyses of stool and shellfish specimens did not reveal a causative agent. Norwalk virus was implicated as the predominant etiologic agent by clinical features of the illness and by seroconversion and the formation of IgM antibody to Norwalk virus in paired serum samples from persons in five (71 percent) of seven outbreaks in which testing was done. In addition, Norwalk virus was identified by radioimmunoassay in clam and oyster specimens from two of the outbreaks. Determining the source of the shellfish was not always possible, but northeastern coastal waters were implicated. The magnitude, persistence, and widespread nature of these outbreaks raise further questions about the safety of consuming raw shellfish.     

Immunoglobulin M responses to the Norwalk virus of gastroenteritis.

Eighty-seven serum specimens from 20 human subjects experimentally inoculated one or more times with Norwalk virus were quantitatively examined for virus-specific immunoglobulin M (IgM). A sensitive and specific radioimmunoassay for anti-Norwalk virus blocking activity was applied to whole serum and to separate IgM and IgG fractions obtained by sucrose density gradient ultracentrifugation. The peak IgM response occurred at about 2 weeks after illness, but IgM was detectable at lower titers for up to 21 weeks after infection. The IgM response was seen in volunteers who became ill, whether or not prechallenge total serum antibody was present. On long-term (27 to 42 months) rechallenge, volunteers who were previously ill and had produced IgM antibody again developed illness, and a secondary IgM response greater than the first was detected. Inoculated volunteers who did not develop illness, as well as previously ill volunteers on short-term rechallenge (4 to 14 weeks), usually failed to generate an IgM response, whether or not an IgG response had occurred. In ill subjects, the rise in IgM and IgG occurred concomitantly. Virus-specific IgM is not necessarily indicative of primary infection with Norwalk agent inasmuch as reinfection produces an enhancement of the IgM response. Furthermore, Norwalk-specific IgM responses do not appear to be associated with subclinical illness.     

Frequency of preclumped virus in routine fecal specimens from patients with acute nonbacterial gastroenteritis

A low-speed centrifugation technique for the preparation of grids after minimal purification of fecal extracts is described for examination of viruses by direct electron microscopy using negative staining. Results showed that adenovirus, astrovirus, rotavirus, and "small round" viruses were frequently shed into the gastrointestinal tract in clumps of variable size. Differential centrifugation study showed that a substantial proportion of the virus in the sample was lost in the initial pellet at the first step of clarification; this finding casts doubt on the validity of immune electron microscopy for direct typing of strains of these viruses from stools. In addition, particle counts based on conventional specimen processing are likely to grossly underestimate the true value.     

Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia.

Three formalin-preserved fecal specimens from the same child attending a child-care center were pooled and compared with the three separate individual specimens by a single microscopic examination of concentration sediment for Giardia lamblia. The sensitivity of the pooled system was 100% when two or more individual specimens were positive and 88% when only one individual specimen was positive. The organism density in a single specimen was not a factor of whether the pool of specimens was positive or negative. Nearly half of the pools that contained positive specimens had only one of three specimens with positive results, reinforcing the need for multiple stool examinations when diagnosing G. lamblia infections.     

PCR assay to detect Bacillus anthracis spores in heat-treated specimens

Recent interest in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. The development of rapid and sensitive techniques to detect anthrax spores in suspicious specimens is the most important aim for public health. With a view to preventing exposure of laboratory workers to viable Bacillus anthracis spores, this study evaluated the suitability of PCR assays for detecting anthrax spores previously inactivated at 121 degrees C for 45 min. The results indicate that heat treatment ensures the complete inactivation of B. anthracis spores without significantly affecting the efficiency of PCR assays.     

A sensitive duplex nanoparticle-assisted PCR assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens

In this study, a novel duplex nanoparticle-assisted polymerase chain reaction (nanoPCR) assay was developed to detect porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis virus (TGEV). Two pairs of primers were designed based on the conserved region within the N gene of PEDV and TGEV. In a screening of 114 clinical samples from four provinces in China for PEDV and TGEV, 48.2 and 3.5 % of the samples, respectively, tested positive. Under optimized conditions, the duplex nanoPCR assay had a detection limit of 7.6 × 10¹ and 8.5 × 10¹ copies μL^?1 for PEDV and TGEV, respectively. The sensitivity of the duplex nanoPCR assay was ten times higher than that of a conventional PCR assay. Moreover, no fragments were amplified when the duplex nanoPCR assay was used to test samples containing other porcine viruses. Our results indicate that the duplex nanoPCR assay described here is useful for the rapid detection of PEDV and TGEV and can be applied in clinical diagnosis.     

Real-time PCR assay to detect smallpox virus

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/ microl to 1 ng/ microl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/ microl to 1 ng/ microl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/ microl to 1 ng/ microl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/ microl to 1 ng/ microl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence.     

Genetic diversity of sapovirus in fecal specimens from infants and children with acute gastroenteritis in Pakistan

Summary. A total of 517 fecal specimens collected from infants and children with acute gastroenteritis in Karachi city, Pakistan during 1990–1994 were examined for the presence of sapovirus by RT-PCR and sequence analysis methods. Sapovirus was identified in 17 of 517 (3.2%) specimens. Sapovirus was further clustered into three distinct genogroups (I, II and IV) and these presented 70.6%, 23.5% and 5.9%, respectively. Our results clearly indicated that sapovirus could be classified into 7 GI and 4 GII genotypes. It was noteworthy to point out that sapovirus detected among Pakistani infants and children with acute gastroenteritis demonstrated the great genetic diversity and presented novel sapovirus genotypes.     

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.     

Issues associated with and recommendations for using PCR to detect outbreaks of pertussis

Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for Bordetella pertussis was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for B. pertussis during both outbreaks, which confirmed that B. pertussis was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that B. pertussis may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for B. pertussis. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease.     

Clinical immunity in acute gastroenteritis caused by Norwalk agent.

To examine immunity in viral gastroenteritis, we challenged and then rechallenged 12 volunteers with Norwalk agent and evaluated symptoms, jejunal biopsies and serum antibody. With the first challenge, gastroenteritis developed in six volunteers but not in the others. When rechallenged 27 to 42 months later, the six who became ill initially again had gastroenteritis with jejunal lesions; in the six previously immune volunteers illness or jejunal lesions did not develop. Four of five ill volunteers had increases in serum antibody to Norwalk agent after both challenges. Serum antibody did not increase in three immune volunteers after either challenge. Four volunteers who had twice become ill underwent a third challenge four to eight weeks after their second illness. In one gastroenteritis developed; in three, it did not. These findings indicate two forms of immunity for viral gastroenteritis, one of short and the other of long duration. Factors other than serum antibody appear important in immunity to Norwalk gastroenteritis.     

Application of indirect immunofluorescence to detection of Dientamoeba fragilis trophozoites in fecal specimens.

An indirect fluorescent-antibody (IFA) assay was carried out to examine for the presence of Dientamoeba fragilis trophozoites in preserved fecal specimens. Antiserum to D. fragilis trophozoites was raised in a rabbit with a dixenic culture of D. fragilis (ATCC 30948) from the American Type Culture Collection. After absorption with Klebsiella pneumoniae and Bacteroides vulgatus, the immune rabbit serum was used for examination by the IFA assay. A total of 155 clinical samples were tested; 42 with no parasites, 9 with D. fragilis, and 104 with other parasites. The IFA assay identified seven D. fragilis organisms. Two specimens with doubtful IFA assay readings showed very scanty amounts of D. fragilis trophozoites on stained smears. There were no false-positive IFA assay readings. The IFA assay appeared to be a promising method because of its speed in screening. The specificity of the IFA assay indicates that other diagnostic tests such as an enzyme-linked immunosorbent assay could be developed to identify D. fragilis antigens in fecal specimens.     

Detection and subsequent sequencing of Puumala virus from human specimens by PCR.

A sensitive method based on PCR was developed for the detection of Puumala virus (PUU) in human samples. The assay was found to be specific for PUU-like strains and distinguished between these and hantaviruses of other serotypes. The detection limit was found to be 10(-5) focus-forming units. Clinical samples were collected from patients with nephropathia epidemica in Sweden and western Russia. Five whole blood samples collected from patients in Russia with the acute phase of disease were found to be positive by the PCR. All samples were negative for PUU antigen when examined by enzyme-linked immunosorbent assay. Virus isolation on Vero E6 cells from several of the acute-phase samples, including the 5 PCR-positive samples, was not successful. The amplified samples were subjected to direct nucleic acid sequencing for confirmation of identity. The sequences differed from each other and were closely related to the Russian bank vole isolate CG-1820, thereby indicating the origin of nephropathia epidemica. The PCR was used for amplification and subsequent nucleotide sequencing of eight PUU-like isolates with different geographic origins. The Swedish strains were more closely related to the Finnish PUU prototype strain, Sotkamo, than to the Russian isolates. Interestingly, a Belgian isolate, CG-13891, differed markedly from all other PUU strains.     

Prevalence of antibody to the Norwalk virus in various countries.

Serum samples from children and adults from several countries were tested by radioimmunoassay for antibody to the Norwalk virus. Antibody was commonly found in adults from all the countries tested. Antibody appears to be acquired more rapidly in children from underdeveloped countries than in children from the United States.