Application of the polymerase chain reaction for the detection of Ehrlichia canis in tissues of dogs

Doxycycline treatment resulted in a carrier status in 3 dogs and complete clearance of Ehrlichia canis in 2 dogs (Iqbal and Rikihisa, 1994). Using specimens obtained during that study applicability of polymerase chain reactions (PCRs) in detecting E. canis DNA in tissue specimens and correlation of PCR results with our previous cell culture isolation results were evaluated. PCRs using a pair of primers specific to E. canis 16SrRNA gene sequence were used to detect DNA of E. canis in tissues of 5 experimentally-infected dogs 2 months after doxycycline treatment. An approximately 600 bp product defined by the specific primers was amplified in blood, kidney, lymph nodes, liver, and/or spleen of 3 dogs from which E. canis was reisolated in cell culture. In contrast, E. canis DNA was not detected in tissue or blood specimens of the 2 dogs from which E. canis was not reisolated after doxycycline treatment or in 2 control uninfected dogs. The findings indicate PCR is effective in detecting E. canis in tissues.     

Reisolation of Ehrlichia canis from blood and tissues of dogs after doxycycline treatment

We present evidence that supports the carrier status of dogs experimentally infected with Ehrlichia canis after treatment with doxycycline. Canine ehrlichiosis was induced in five dogs by intravenous inoculation with E. canis-infected DH82 cells. All animals developed mild clinical signs of transient fever, body weight loss, thrombocytopenia, and increased gamma globulin levels in plasma. An indirect fluorescent-antibody test (IFA) revealed that all dogs had seroconverted (titer, 5,120) by day 10 postinoculation (p.i.). E. canis was reisolated from blood samples collected at intervals throughout the 2-month period p.i. Doxycycline was administered orally once daily at 10 mg/kg of body weight per day for 1 week starting at 2 months p.i. Following treatment, gamma globulin levels in plasma were decreased. At necropsy on days 54 to 59 after the start of treatment, spleen, liver, kidney, and lymph nodes were collected for E. canis culture and histopathologic examination. Although the dogs did not show significant clinical signs during or after treatment with the antibiotic, E. canis was reisolated from the blood and tissue samples of three of five dogs. A 16-fold reduction in IFA titer was noted in two dogs which were negative for E. canis reisolation at day 49 after the start of treatment, whereas a zero- to fourfold reduction in IFA titer was seen in the remaining three dogs. Western immunoblot reactions to higher-molecular-size E. canis antigens in the sera of two dogs which were negative for E. canis on blood culture decreased, whereas they remained continuously high or only transiently decreased for the duration of the study for antigens in the sera of three dogs from which E. canis was reisolated. Histopathologically, prominent plasmacytosis in the kidney cortex was present in three dogs from which E. canis was reisolated, whereas the kidney cortices of two dogs had moderate to minor plasmacytosis. These findings pose questions regarding the efficacy, dosage and duration of doxycycline treatment in dogs with E. canis infection. In addition, it was shown that IFA and Western immunoblotting may aid in assessing the efficacy of antibiotic therapy when definitive reisolation procedures are not readily available.     

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.     

Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland

Serum samples from 996 dogs in Switzerland were examined for antibodies to Ehrlichia canis and to the agent causing canine granulocytic ehrlichiosis (CGE). Ehrlichiosis, borreliosis, and systemic illness not associated with ticks were suspected in 75, 122, and 157 of these dogs, respectively. The remainder of the serum samples were obtained from clinically healthy dogs which resided north (n = 235) or south (n = 407) of the Alps. The serum samples were tested by an indirect immunofluorescence technique for antibodies to the two agents incriminated, E. canis and Ehrlichia phagocytophila, a surrogate marker of the agent of CGE. Twenty-two of 996 (2.2%) serum samples had antibodies to E. canis and were distributed as follows: 20 of 75 (26.7%) samples from dogs suspected of having ehrlichiosis, 1 of 122 (0.8%) from dogs suspected of having borreliosis, and 1 of 407 (0.2%) from healthy dogs which resided south of the Alps. Of the 75 (7.5%) serum samples that had antibodies to E. phagocytophila, significantly more samples were from ill dogs than from healthy dogs. Among the sera from healthy dogs, antibodies to E. phagocytophila were significantly more prevalent in the north. Because seropositive dogs had a history of travel outside Switzerland and because Rhipicephalus sanguineus is found exclusively south of the Alps, it was presumed that, in contrast to the agent of CGE, E. canis is not indigenous to Switzerland.     

A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle

A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of granulocytes containing ehrlichiae (morulae). Thirty-four of 36 microscopically positive samples were also positive by PCR, and 6 microscopically negative samples were negative by PCR as well. Six samples, in which morulae-like structures had been seen, were negative by PCR, also at a lower annealing temperature and when a reamplification of the first PCR products was performed. The identities of the PCR products from some canine and equine isolates were verified by direct DNA sequencing and were found to be identical with the Ehrlichia sequence found in these animal species that had been obtained earlier. The sequences of a segment of approximately 600 nucleotides from two bovine isolates were identical to that of E. phagocytophila, whereas the sequence of another bovine isolate differed in two positions from that of E. phagocytophila and in three positions from the sequences of the canine and equine isolates. Serum samples were analyzed by indirect fluorescent-antibody testing. Seventy-three percent of the animals which were positive by microscopy and PCR also had positive antibody titers. However, it was not possible to rely on a single serological result for diagnosis of present infection. It was, therefore, concluded that PCR was the most reliable method, useful in the clinical laboratory for specific and early diagnosis of granulocytic ehrlichiosis in animals.     

Granulocytic ehrlichiosis in two dogs in Switzerland

This case report describes two dogs with granulocytic ehrlichiosis. Dog 1 was a male Labrador retriever with clinical signs of lymphosarcoma. Dog 2 was a female Airedale terrier, whose clinical signs included apathy, pyrexia, diarrhea, and abdominal pain. Examination of blood smears revealed Ehrlichia organisms in the neutrophils of both dogs. There was thrombocytopenia in both dogs, and dog 2 also had leukopenia. In both dogs, bands of identical length were amplified from DNA of leukocytes via nested PCR. The bands had identical nucleotide sequences, which differed from the gene sequences of Ehrlichia equi and E. phagocytophila in three and two positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% homologous to that of a human granulocytic ehrlichia.     

Serologic and molecular detection of granulocytic ehrlichiosis in Rhode Island

A new indirect fluorescent-antibody (IFA) assay with antigen produced in vitro in the human promyelocytic leukemia cell line HL60 was used to identify the first recognized case of human granulocytic ehrlichiosis in Rhode Island. This IFA assay was used to detect granulocytic ehrlichiae in white-footed mice and in a dog inhabiting the area surrounding the patient's residence. Host-seeking Ixodes scapularis ticks found in the same habitat also were infected. I. scapularis ticks collected from other locations were fed on dogs and New Zealand White rabbits to assess the competency of these species as hosts of granulocytotropic Ehrlichia. Tick-induced infections of dogs were confirmed by serologic testing, tissue culture isolation, and PCR amplification, whereas several rabbits seroconverted but were PCR and culture negative. PCR amplification of the 16S rRNA gene and DNA sequencing of the PCR products or culture isolation was used to confirm granulocytic Ehrlichia infections in humans, dogs, white-footed mice, and ticks.     

Hemorrhage in cavernous hemangioma of the adrenal gland: US, CT and MRI appearances with pathologic correlation

A total of 374 dogs, 252 from five military kennels and 122 privately owned, were tested for Ehrlichia canis antibody. Sera were tested at a 1:20 dilution by indirect fluorescent antibody with the use of E. canis cell-culture antigen slides. The overall prevalence of E. canis antibody was 33%. Antibody prevalence among military dogs (29%) was significantly lower than among privately owned dogs (41%; P < 0.05). The E. canis seroprevalence among dogs infested with ticks (Rhipicephalus sanguineus) was higher (44%) than that among uninfested dogs (31%; P = 0.08). The seroprevalence among military dogs varied from 21-46% at the five kennels; lower prevalences were observed in kennels with higher sanitary and hygienic conditions. Age- and sex-related E. canis antibody prevalences were not significantly different among military and privately owned dogs, although adult and male privately owned dogs had the highest seroprevalences (45% and 44%, respectively). Three dogs with epistaxis had E. canis antibody titres > 1:320. These data demonstrate the first laboratory evidence of E. canis infection among dogs in Egypt.     

Serological responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in patients from New York State

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.     

Survey of tick-borne diseases in dogs infested with Rhipicephalus sanguineus at a kennel in Okayama Prefecture, Japan

Serological tests were performed to investigate extent of tick-borne diseases in dogs infested with Rhipicephalus sanguineus at a kennel in Okayama Prefecture. Three of 22 dogs (13.6%) were positive for Ehrlichia canis. Two of 19 dogs (10.5%) were positive for Rickettsia japonica. Three of 22 dogs (13.6%) were positive for Babesia gibsoni. None of these animals were positive for Coxiella burnetii or Hepatozoon canis. A microfilaria was detected in a drop smear of hemolymph from an engorged female tick, however, species was not determined. It is possible that these ticks can transmit pathogens to domestic dogs which are rare in Japan.     

Identification of a granulocytic Ehrlichia strain isolated from a horse in Switzerland and comparison with other rickettsiae of the Ehrlichia phagocytophila genogroup

This case report describes a 12-year-old Arabian mare with granulocytic ehrlichiosis. Clinical signs included fever, apathy, anorexia, icterus, limb edema, and reluctance to move. Examination of buffy coat smears revealed Ehrlichia organisms in neutrophils and eosinophils. A band of 1,428 bp was amplified from DNA of leukocytes via nested PCR and was identified as part of the Ehrlichia 16S rRNA gene. It differed from the gene sequences of Ehrlichia phagocytophila and E. equi at two and three positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% identical to that of the agent of human granulocytic ehrlichiosis.     

Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp. , p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.     

Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

Kinetics of serologic cross-reactions between Ehrlichia canis and the Ehrlichia phagocytophila genogroups in experimental E. canis infection in dogs

The serological cross-reactions between the Ehrlichia canis and Ehrlichia phagocytophila genogroups, and the kinetics of development of antibodies in dogs to the E. phagocytophila group after artificial infection with the Israeli strain of E. canis was investigated. Results of this study indicate that the development of antibodies to the E. phagocytophila genogroup in dogs after infection with E. canis is a time dependent event probably conditional on the continued propagation of the rickettsia in the host. After spontaneous clinical recovery, and at the beginning of the subclinical phase, no antibodies to E. phagocytophila were yet detectable. The first evidence of IgG antibodies to E. phagocytophila were found in two of the six dogs, 55 days after artificial infection with E. canis, while another two dogs became seropositive to E. phagocytophila 22 days later. All surviving dogs were seropositive to E. phagocytophila by 150 days PI. It is suggested that the appearance of E. phagocytophila antibodies following treatment of acute E. canis infection may be used to judge treatment failure and/or persistence of infection.     

Experimental infection of four horses with Ehrlichia phagocytophila

Four clinically healthy horses which were negative for antibodies to Ehrlichia phagocytophila, the agent of bovine ehrlichiosis, were infected experimentally with E phagocytophila-containing bovine leucocytes, administered intravenously. The horses were examined daily for four weeks, and blood samples were collected daily for cytological, haematological and biochemical examination and for a nested polymerase chain reaction (PCR). An indirect immunofluorescence test was used to determine when the horses seroconverted and the duration of positive titres. There were no abnormal clinical, haematological or biochemical findings in any of the four horses and all the PCRs yielded negative results. However, all the horses seroconverted with reciprocal titres of up to 1280, and the positive titres persisted for up to five months.     

Changes in blood sugar and ammonium levels after ligation of the afferent hepatic vessels

Certain aspects of the development of Ehrlichia canis, causative agent of canine ehrlichiosis (tropical canine pancytopenia) in Rhipicephalus sanguineus ticks were studied. It was found that partial feeding of nymphs infected as larvae with E canis was a desirable, if not necessary, preliminary treatment for successful infection of dogs with ground-up ticks. It remains unclear whether feeding increased the number or altered the virulence of ehrlichiae within tick tissues. Ehrlichia canis organisms were detected by immunofluorescent microscopy in the midgut and hemocytes and by electron microscopy in the midgut and salivary glands of partially engorged adult ticks which had been infected as larvae and nymphs. Organisms were not observed in the ovary. Intracytoplasmic inclusions contained 1 to 80 elementary bodies, each provided with 2 distinct membranes. Infection of the midgut and salivary gland was confirmed by injecting homogenates of these tissues into susceptible dogs. Staining of gut smears of partially engorged adult ticks by fluorescein-conjugated anti-E canis antibody was found to be a reliable indicator of the infection.     

How many measurements are necessary in diagnosing mild to moderate hypertension?

Cowdria ruminantium is the etiologic agent of heartwater, a tick-transmitted foreign animal disease with considerable potential for entrance into the USA. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect serologic responses to C. ruminantium infection. The cELISA utilized a recombinant form of the C. ruminantium major antigenic protein (MAP-1) as the antigen and an anti-MAP-1 monoclonal antibody as the competing indicator reagent. Experimental antisera to C. ruminantium and a wide variety of related ehrlichial organisms were used to evaluate cELISA reactivity. Only sera against C. ruminantium, Ehrlichia canis, E. chaffeensis, and a recently discovered cervine ehrlichia-like organism reacted positively in the cELISA. Specificity of the cELISA was > or = 99.5% in a survey of 1,774 southeastern US and Puerto Rican slaughter cattle sera but was only 85% in a group of 79 hunter-killed white-tailed deer (Odocoileus virginianus) from the southeastern USA. Reference true-positive and cELISA false-positive sera were further analyzed by end point titrations using the cELISA and by indirect fluorescent antibody (IFA) tests for reactivity with C. ruminantium, E. canis, and E. chaffeensis antigens. True heartwater-positive sera were significantly more reactive using the cELISA and C. ruminantium IFA procedures (P < 0.05), whereas false-positive sera were significantly more reactive with the antigens used in the E. chaffeensis IFA procedure (P < 0.05). A group of sera from 210 field-origin ruminants residing on known or potentially heartwater-endemic Caribbean islands revealed a substantial (12.4%) prevalence of cELISA-positive specimens. The cELISA is a relatively specific serodiagnostic test for heartwater in cattle and could be used to monitor for possible introduction of the disease into the USA. The cELISA may also be an excellent tool for monitoring the success of an ongoing Caribbean Amblyomma tick eradication program designed to eliminate the biological vector responsible for the perpetuation and spread of this dangerous foreign animal disease.     

Experimental transmission of Ehrlichia canis (Rickettsiales: Ehrlichieae) by Dermacentor variabilis (Acari: Ixodidae)

Four trials were conducted in which laboratory-reared Dermacentor variabilis nymphs were exposed to Ehrlichia canis by feeding on experimentally infected dogs as soon as classical morulae were detected in peripheral blood monocytes. After molting 25, 50 or 90 adult tick pairs were permitted to feed on 7 Ehrlichia-naive dogs. Transmission occurred in trials 1 (1/1 dog), 3 (1/1 dog) and 4 (2/2 dogs) but not in trial 2 (0/3 dogs), with 4 of 7 dogs becoming infected. Successful transstadial transmission was demonstrated by detection of morulae in peripheral blood lymphocytes and by seroconversion to Ehrlichia canis 30 d post-exposure. Incubation periods ranged between 17 and 22 days (mean = 19). Clinical signs, typical of ehrlichiosis, included mucopurulent ocular discharge, lymphadenopathy and malaise with accompanying pyrexia, leukopenia and thrombocytopenia. Pyrexia, thrombocytopenia and erythrophagocytosis and vacuolization of the cytoplasm of monocytic cells were observed 1-4 d prior to detection of morulae. This is the first demonstration that a tick other than Rhipicephalus sanguineus is capable of transstadial transmission of this important pathogen of dogs.     

Detection of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Pleuroceridae: Juga spp.) from northern California

Ehrlichia DNA was identified by nested PCR in operculate snails (Pleuroceridae: Juga spp.) collected from stream water in a northern California pasture in which Potomac horse fever (PHF) is enzootic. Sequencing of PCR-amplified DNA from a suite of genes (the 16S rRNA, groESL heat shock operon, 51-kDa major antigen genes) indicated that the source organism closely resembled Ehrlichia risticii, the causative agent of PHF. The minimum percentage of Juga spp. harboring the organism in the population studied was 3.5% (2 of 57 snails). No ehrlichia DNA was found in tissues of 123 lymnaeid, physid, and planorbid snails collected at the same site. These data suggest that pleurocerid stream snails may play a role in the life cycle of E. risticii in northern California.     

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.