The idenficaton of apparently fastidious microorganisms is often problematic. DNA from a rickettsialike agent (called the ELB agent) present in cat fleas could be amplified by PCR with conserved primers derived from rickettsial 17-kDa common protein antigen and citrate synthase genes but not spotted fever group 190-kDa antigen gene. Alm I sites in both the 17-kDa and citrate synthase PCR products obtained with the rickettsia-like agent and Rickkesia lyphi were different even though both agents reacted with monoclonal antibodies previously thought specific for R. typhi. The DNA sequence of a portion of the 17-kDa PCR product of the rickettsia-like agent differed significantly from all known rickettsial sequences and resembled the 17-kDa sequences oftyphus more than spotted fever group rickettsiae. The rare stable transovarial maintenance of this rickettsia in cat fleas has important implications for the disease potential of cat fleas.

Hepatitis E virus is responsible for both sporadic and epidemic hepatitis in developing countries. The nonenveloped virus is 27-34 nm in diameter and has been shown to contain a single-strand, positive-sense, polyadenylylated RNA genome of -7.5 kilobases. The nucleotide sequence of the Burma strain of hepatitis E virus has been reported and three open reading frames (ORFs) have been identified. The deduced amino acid sequence from each of these ORFs was used to synthesize overlapping peptides (decamers overlapping at every fourth amino acid) on a solid phase. These peptides were then tested in an ELISA with pooled acute-phase sera from known cases of enterically transmitted non-A, non-B hepatitis collected in the Sudan. Linear B-cell epitopes were identified in all three ORFs. Epitopes were identified throughout the polyprotein encoded by ORF1, but they appeared to be particularly concentrated in the region of the RNA-dependent RNA polymerase. Distinct epitopes were identified in the presumed structural protein encoded by ORF2, and one epitope was identified dose to the carboxyl terminus of the protein encoded by ORF3. These data precisely pinpoint linear B-cell epitopes recognized by antibodies from patients with acute hepatitis E and identify an antibody response directed against the RNA-dependent RNA polymerase.A major form of non-A, non-B hepatitis is the enterically transmitted (ET-NANB) form that is known to frequently occur in developing countries. Outbreaks of ET-NANB hepatitis in Asia (1-3), USSR (4), Costa Rica and Mexico (5), and several African nations (6) have generally been traced to contaminated water (7,8). Males and females appear to be equally affected, and the clinical course is similar to that of classic hepatitis A except for a high mortality rate (10-20%o) in pregnant women (9, 10). There is no evidence of chronic hepatitis following acute ET-NANB hepatitis (11).Virus-like particles with a diameter of 27-34 nm have been recovered from the stools of patients with ET-NANB hepatitis (10-12). The viral agent has been serially transmitted in cynomolgus macaques (13-15), and a partial cDNA clone from the responsible virus was isolated from infected cynomolgus bile (15). The hepatitis E virus (HEV) appears to be distantly related to known positive-strand RNA viruses (16) and has a genome of -7.5 kilobases (kb) that contains three open reading frames (ORFs) encoding viral proteins (17). ORF1, the largest of the three, extends -5 kb from the 5' end. ORF2, the next largest, begins 37 base pairs (bp) downstream of ORF1 and extends "2 kb to the termination codon present 65 bp from the 3'-terminal stretch of adenosine residues. ORF3 partially overlaps ORF1 and ORF2 and contains only 369 bp (17). ORF1 is believed to encode nonstructural proteins including an RNA-dependent RNA polymerase (RDRP) (15, 16) and a helicase-like region believed to promote the unwinding of DNA-RNA duplexes, which are required for genome replication, recombination, repair, and transcription of viral genes (16). ORF2 en...

Polymerase chain reaction (PCR) amplification of DNA was used to detect the etiologic agent of murine typhus, Rickettsia typhi, in experimentally infected adult fleas. A primer pair derived from the 17-kilodalton antigen sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base-pair (bp) fragment of the genome of the murine typhus rickettsiae. The amplified 17-kilodalton protein antigen-specific sequence was detected in ethidium bromide-stained agarose gels in individual fleas as early as 2 days after exposure to rickettsemic rats (two of six tested). The 434-bp sequence was not detected in uninfected control fleas. A dot hybridization assay used to detect the 434-bp fragment was also specific and about 100-fold more sensitive than the agarose gel PCR assay. Since the PCR assay employed a boiled extract of triturated fleas, both PCR and an antigen capture enzyme-linked immunosorbent assay (ELISA) could be performed on the same individual flea homogenate. The ELISA identified 12 infected fleas out of 29 randomly selected fleas, compared with 14 specimens which were positive by PCR. The PCR assay detected rickettsiae in samples in which no viable rickettsiae were detected by plaque assay. Like the ELISA, the PCR assay sensitivity was due in part to its suitability for detecting small numbers of both live and dead R. typhi in fleas.

A newly developed Western blot assay for antibody to hepatitis E virus (anti-HEV) was used to evaluate 39 cases of acute pediatric hepatitis and 39 control patients in Khartoum, Sudan. The mean age of cases was 6.5 years (range, 2-14); 64% were male. Acute hepatitis A (IgM anti-HAV-positive) was diagnosed in 13 cases, acute hepatitis B (IgM anti-HBc-positive) in 1, and acute hepatitis E (positive for IgM anti-HEV) in 23 (59%). None of the cases with IgM anti-HAV or IgM anti-HBc had IgM anti-HEV; 3 controls had IgM anti-HEV. Acute hepatitis E was associated with recent contact with a family member or acquaintance with jaundice and the presence of indoor plumbing. The newly developed hepatitis E assay appeared to be specific for the diagnosis of acute icteric non-A, non-B hepatitis. Hepatitis E was found to be the most common cause of acute sporadic hepatitis in children living in an urban area of Africa.

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The DNA sequence of the gene encoding the protective surface protein antigen (SPA) of Rickettsia prowazekii has been determined. The open reading frame of 4836 nucleotides with promoter and ribosome-binding site is present on a 10.1-kilobase EcoRI fragment. The encoded carboxyl terminus of the 169-kDa protein contains a potential transmembrane region and hydrophilic regions with many lysine and arginine residues potentially accessible to proteolytic cleavage. Because the rickettsia-derived SPA has an estimated molecular mass of only 120 kDa and does not contain several predicted large carboxyl-region CNBr fragments, the SPA product appears to be processed by the rickettsiae. Eight other CNBr fragments were identical in sequence to those predicted from the encoded gene. A complementary 8.7-kilobase EcoRI fragment of Rickettsia typhi DNA was cloned. This fragment lacked a 1433-base-pair region that included the promoter, ribosome-binding site, and the initial 1162 base pairs of the open reading frame encoding the R. prowazekii SPA but had a 3674-base-pair region identical with the remainder of the R. prowazekii SPA gene sequence.

A cross-sectional study was undertaken to determine the prevalence of Mycoplasma fermentans infection in patients with human immunodeficiency virus (HIV) infection using polymerase chain reaction methodology. Targeted M. fermentans DNA sequences could be amplified from the DNA extracted from the blood of 6 (11%) of 55 HIV-seropositive patients but from none of 26 HIV-seronegative subjects at low risk for HIV infection (P = .17). There was no correlation between M. fermentans infection and HIV clinical stage. There was a nonsignificant trend toward an association between M. fermentans infection and a history of syphilis. Infection with M. fermentans may occur more commonly in HIV-infected patients; however, a role as a copathogen or opportunistic infection was not established in this study.

Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis. The virulent strains of B. anthracis are encapsulated and toxigenic. In this paper we describe the development of a PCR technique for identifying spores of B. anthracis. Two 20-mer oligonucleotide primers specific for the capB region of 60-MDa plasmid pXO2 were used for amplification. The amplification products were detected by using biotin-and fluorescein-labeled probes in a novel dual-probe hybridization format. Using the combination of PCR amplification and dual-probe hybridization, we detected two copies of the bacterial genome. Because the PCR assay could detect a minimum of 100 unprocessed spores per PCR mixture, we attempted to facilitate the release of DNA by comparing the effect of limited spore germination with the effect of mechanical spore disruption prior to PCR amplification. The two methods were equally effective and allowed us to identify single spores of B. anthracis in PCR mixtures.