Molecular epidemiology: applications of nucleic acid amplification and sequence analysis.
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Biomedical subjects
Publications and source records attributed to J E McDade.
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Epidemic, louse-borne typhus persists in the rugged, mountainous areas of Ethiopia and much of northeastern and central Africa as well as in the rural highlands of Central and South America, where the conditions of living favor the harboring of body lice and where antibiotic treatment and effective louse-control measures are unavailable. The historical significance and current epidemiology of typhus, including the reservoir of Rickettsia prowazekii in flying squirrels in the United States, are reviewed, and the clinical presentation, laboratory findings, and hospital course in the cases of 60 patients admitted with epidemic, louse-borne typhus to the St. Paul's Hospital in Addis Ababa, Ethiopia, are described. Treatment of this disease with oral doxycycline, tetracycline, or chloramphenicol prevents complications and results in prompt resolution of symptoms.
Twenty-six cases of a spotted-fever-like illness have been identified on Flinders Island, Tasmania, over a 17 year period. These patients and 335 healthy persons from the island were investigated serologically using the Weil-Felix agglutination test (Proteus sp. antigens OX2, OX19, OXK) and rickettsia-specific microimmunofluorescence. The antigens used in these latter tests comprised one member of the typhus group (Rickettsia typhi) and three members of the spotted fever group (Rickettsia rickettsii, Rickettsia australis and Rickettsia conorii). Patients with Flinders Island spotted fever showed a higher prevalence of positive reactions to the Weil-Felix tests (with OX2 and OX19 antigens) and a higher prevalence of positive results to rickettsia-specific serological tests (with the exception of antibodies to Rickettsia typhi) than did healthy persons; OX2 (36% v. less than 1%); OX19 (36% v. less than 1%); Rickettsia rickettsii (42% v. 1%); Rickettsia australis (46% v. 1%); Rickettsia conorii (42% v. 1%); Rickettsia typhi (4% v. 4%). In seven of the 26 patients (27%) seroconversion was demonstrated by means of Weil-Felix tests, confirming recent infection. In six of these patients seroconversion was also demonstrated in rickettsia-specific tests. Although these results support the clinical evidence that the illness on Flinders Island is caused by a rickettsia of the spotted fever group, the aetiological agent remains to be isolated.
Rickettsioses have nonspecific clinical manifestations, making them difficult to diagnose in a clinical setting. Laboratory testing is usually needed to confirm the diagnosis. Rickettsial isolation is a sensitive and specific diagnostic technique, but the hazards associated with handling pathogenic rickettsiae usually preclude isolation attempts in most laboratories. Rickettsiae can also be detected in infected tissues by fluorescein-labeled antisera or by immunoperoxidase staining, but these techniques lack sensitivity, except when applied to postmortem tissue specimens. However, rickettsial DNA can be detected in acute phase blood specimens by polymerase chain reaction (PCR) technology, and this technique offers the prospect of prompt diagnosis and treatment. Serologic testing remains the most frequently used approach to diagnosis, although antibody tests usually fail to identify rickettsioses early enough to affect the management of individual patients. Available serologic techniques vary considerably in their sensitivity and specificity. Enzyme-linked immunosorbent assays (ELISA) are extremely sensitive, but the general unavailability of specific diagnostic antigens reduces the specificity of this and other serologic techniques. Molecular characterization of rickettsial antigens may soon allow the production of peptide antigens that are specific for each species and could maximize the specificity of test results. No diagnostic technique has any value unless it is applied successfully to the appropriate patient population. Improved surveillance of rickettsial diseases is urgently needed to identify specific areas in which rickettsioses are endemic. Such surveillance data would promote awareness of rickettsioses among local physicians and increase the probability that individual patients with rickettsioses would be identified promptly and receive appropriate therapy early in the course of their illness.
Ehrlichiae are one of several kinds of obligate intracellular bacteria. Taxonomically, they are grouped with rickettsiae, but they can be distinguished by their unique tropism for circulating leukocytes. Ehrlichia canis causes a pancytopenia in dogs that becomes chronic if untreated. Certain breeds develop severe infections, characterized by fever, anorexia, dramatic weight loss, marked pancytopenia, anemia, peripheral edema, and hemorrhage. Ehrlichia risticii, a recently discovered species, is the cause of a serious diarrheal disease of horses. Other species of ehrlichiae have been documented as being veterinary pathogens. Recent data indicate that E. canis or a closely related species causes an acute febrile illness in humans. Clinically, the disease is similar to Rocky Mountain spotted fever, except that most patients do not have a rash. Human ehrlichiosis appears to be tickborne and is prevalent primarily in the southern Atlantic and south-central states. A mild from of ehrlichiosis has also been documented.
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A polymerase chain reaction (PCR) procedure for detecting rickettsial DNA was developed and shown to be specific for Rickettsia rickettsii and R. conorii, the etiologic agents of Rocky Mountain spotted fever (RMSF) and Boutonneuse fever, respectively. Blood clots were obtained from nine confirmed RMSF patients and six controls and analyzed for the presence of rickettsial DNA by the PCR method. A defined region of the rickettsial genome was successfully amplified from seven of the nine clinical specimens tested; all six control specimens gave negative results. These findings indicate that R. rickettsii can be detected early after the onset of RMSF, possibly facilitating the decision regarding appropriate antibiotic therapy for some patients. Further refinement of PCR technology could make this procedure a mainstay in the clinical laboratory.
PURPOSE AND METHODS: The rickettsioses continue to constitute major health problems in many parts of the world. With increasing international travel, recognition of rickettsial diseases by physicians is becoming more important. The clinical features of four cases of rickettsial disease imported into Canada over a five-year period are presented; two patients with tick typhus (Rickettsia conorii), one patient with scrub typhus (R. tsutsugamushi), and one patient with murine typhus (R. typhi). We also present the North American data over the past 10 years from the Centers for Disease Control (CDC) (Atlanta). RESULTS: Since 1983 in the United States, three cases of imported scrub typhus, all after travel to India, were confirmed, as well as six cases of murine typhus after travel to southeast Asia. At the CDC, 67 imported cases of tick typhus have been confirmed by indirect fluorescent antibody test since 1976; most illnesses occurred after travel to Africa. CONCLUSION: Rickettsial diseases are underrecognized by physicians, who should consider these diagnoses in travelers returning from endemic areas. Since effective treatment is available, prompt diagnosis and treatment are important. In all cases, specific serologic confirmation should be obtained.
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Monoclonal antibodies were produced from mice infected with Rickettsia akari (the etiologic agent of rickettsialpox) and evaluated for specificity in indirect fluorescent-antibody tests with 23 different rickettsial antigens. Of the nine antibodies that were evaluated, two were specific for R. akari and four reacted with R. akari and all other spotted fever group rickettsiae. The remaining three antibodies reacted with some, but not all, members of the spotted fever group. None of the antibodies reacted with typhus, scrub typhus, trench fever, or Q fever rickettsiae. Adding these antibodies to the list of available diagnostic reagents will facilitate identification of rickettsial diseases, particularly those caused by members of the spotted fever group, where the clinical presentations are similar and the etiologic agents are closely related antigenically.
The Ehrlichia are tick-borne rickettsial organisms that cause disease in animals throughout the world but that have been previously recognized as human pathogens only in Asia. We have identified six patients with serological evidence of recent infection with an Ehrlichia: a fourfold or greater rise or fall in titer to Ehrlichia canis. All of the patients reported recent tick bites. Rigors, myalgia, headache, nausea, and anorexia were each reported by five patients. Fever was present in all patients and was accompanied by relative bradycardia and leukopenia in five patients, thrombocytopenia and abnormal liver function test results in four, and anemia in three. Five of the six patients were treated with tetracycline hydrochloride, and all recovered. Infection with Ehrlichia should be considered in patients with unexplained febrile illnesses after tick exposure.
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DNA obtained from the Sheila Smith strain of Rickettsia rickettsii was digested to completion with the restriction endonucleases BamHI and SalI and ligated with the plasmid vector pUC19. The ligation mixture was used to transform Escherichia coli. A total of 465 bacterial clones were screened for antigen production with hyperimmune rabbit serum. One of the reactive clones, containing a recombinant plasmid designated pSS124, was solubilized and subjected to immunoblot analysis and revealed expression of a 17-kilodalton protein reactive with anti-R. rickettsii serum that comigrated with an antigen from R. rickettsii. A 1.6-kilobase PstI-BamHI fragment from pSS124 was subcloned and continued to direct synthesis of the 17-kilodalton antigen. The nucleotide sequence was determined for this 1.6-kilobase subclone, which encompassed the gene encoding the polypeptide as well as flanking regions containing potential regulatory sequences. The open reading frame consisted of 477 nucleotides that specified a 159-amino-acid protein with a calculated molecular weight of 16,840. The deduced amino acid sequence contained a hydrophobic sequence near the amino terminus that resembled signal peptides described for E. coli. The carboxy terminus was hydrophilic in nature and probably contained the exposed epitopes.
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A serosurvey for evidence of rickettsial infections was conducted in the rural populations of several tropical rain forest areas in Sierra Leone and Ivory Coast. Seropositivity rates were surprisingly high in both countries, with more than 7% of the individuals in some districts having antibodies to spotted fever-group rickettsiae. No significant difference was found in the overall prevalence of diagnostic antibody titers to spotted fever-group rickettsiae in Sierra Leone (5.3%) and Ivory Coast (6.2%). However, there was a significant difference (p less than 0.001) in the prevalence of diagnostic antibody titers to typhus rickettsiae in the two countries. There were no marked geographic differences within either country in overall prevalence of rickettsial infections, but there were possible area differences in specific seropositivity rates to typhus- and spotted fever-group rickettsiae in Sierra Leone. In both countries, age and sex differences were important in determining seropositivity, but there was no indication of an age-sex interaction. In Sierra Leone, 59 of the 80 positive sera (73.8%) were from persons age 15 or above (p less than 0.001), and 50 of the 80 (62.5%) were from males (p = 0.05). In Ivory Coast, 33 of the 37 positive sera (89.2%) were from the greater than or equal to 15-age group, and 28 of the 37 (75.7%) were from males (p less than 0.001 for both age and sex). The identification of specific areas endemic for these rickettsial diseases should facilitate the diagnosis and treatment of patients with rickettsial illnesses in West Africa.