Human immunodeficiency virus (HIV)-positive women may represent one of the fastest-growing populations at risk for acquiring cervical cancer and thus require frequent screening. The purpose of the present studies was to validate a PCR-based urine assay by comparing detection and genotyping of human papillomavirus (HPV) DNA in urine samples and matching cervical swab specimens of HIV-positive women. Despite a difference in amplifiability, the prevalence of any HPV genotype (58% for the cervical swab specimens and 48% for the urine specimens) was not significantly different in this population. The levels of concordance were 70, 71, and 78% for detection of any HPV type, any high-risk HPV type, or any low-risk HPV type in the two specimen types, respectively. While instances of discordant detection were greater for the cervical swab specimens than for the urine specimens, this was not statistically significant. The distributions of HPV genotypes were similar in the cervix and the urine for the majority of types examined. Importantly, detection of HPV DNA in urine was associated with an abnormal Papanicolaou smear to the same extent that detection of HPV DNA in a cervical swab specimen was. These data provide preliminary support for the proposal to use urine testing as a primary or secondary screening tool for cervical cancer in HIV-positive women or as an epidemiological tool. Additional studies with larger sample sizes must be conducted in order to further verify these findings. (25). Importantly, infection with HPV, even high-risk types, is asymptomatic in most people and usually does not lead to cancer. However, more than 35 HPV types have been found to be associated with at least 90% of cases of cervical intraepithelial neoplasia, which is a precursor lesion to cervical cancer (16,21).Although the medical standard for the diagnosis of HPV disease is the Papanicolaou (Pap) smear, screening by this type of method has inherent problems. Only 15 to 50% of patients with HPV infections are accurately identified by Pap smears (22,26). Additionally, the efficacy of screening by use of the Pap smear relies on repeated (yearly) visits. Approximately 10% of women in the United States have never had a Pap smear, and about 30% of women do not have them on a regular basis (1,2,3,29). Furthermore, Pap smear screening requires a pelvic examination, which is invasive and uncomfortable for the patient as well as time-consuming for the health care provider (27). Perhaps the use of urine sampling for routine detection of HPV could provide a preliminary screen for cervical cancer and thus circumvent the need for an annual Pap smear for women who are negative for HPV DNA. Alternatively, detection of HPV DNA in urine could possibly function as a secondary screening technique for cervical cancer in that it could be used to triage women with atypical squamous cells of undetermined significance (ASCUS).Methods for the detection of DNA in patient urine have recently been used to diagnose other common STDs that affect the cervix, such as...

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The diagnoses of Lyme disease based on clinical manifestations, serological findings and detection of infectious agents often contradict each other. We tested 52 blind-coded serum samples, including 20 pre-treatment and 12 post-treatment sera from clinically suspect Lyme disease patients, for the presence of residual Lyme disease infectious agents, using nested PCR amplification of a signature segment of the borrelial 16S ribosomal RNA gene for detection and direct DNA sequencing of the PCR amplicon for molecular validation. These archived sera were split from the samples drawn for the 2-tier serology tests performed by a CDC-approved laboratory, and are used as reference materials for evaluating new diagnostic reagents. Of the 12 post-treatment serum samples, we found DNA evidence of a novel borrelia of uncertain significance in one, which was also positive for the 2-tier serology test. The rest of the post-treatment sera and all 20 control sera were PCR-negative. Of the 20 pre-treatment sera from clinically suspect early Lyme disease patients, we found Borrelia miyamotoi in one which was 2-tier serology-negative, and a Borrelia burgdorferi in two—one negative and one positive for 2-tier serology. We conclude that a sensitive and reliable DNA-based test is needed to support the diagnosis of Lyme disease and Lyme disease-like borreliosis.

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The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing. A 50-base sequence excised randomly from the sequencing electrophoretogram between the 2 nested PCR primer binding sites was sufficient for the Basic Local Alignment Search Tool (BLAST) analysis to validate the B burgdorferi sensu lato 16S rDNA without a reasonable doubt. Nested PCR increased the sensitivity of DNA detection by 100- to 1,000-fold. DNA sequence validation based on BLAST algorithms using the GenBank database practically eliminates any possibility of false-positive results due to molecular misidentification. This technology may be a valuable supplement to the current serologic tests for Lyme disease.

The newly gained knowledge of the viral etiology in cervical carcinogenesis has prompted industrial interests in developing virology-based tools for cervical cancer prevention. Due to the long incubation period from viral infection to developing an invasive cancer, a process whose outcome is influenced by numerous life-style and genetic factors, the true efficacy of the genotype-specific human papillomavirus (HPV) vaccines in cervical cancer prevention cannot be determined for another 30 years. Most HPV DNA test kits designed to replace the traditional Papanicolaou (Pap) smears for precancer detection lack the analytical sensitivity and specificity to comprehensively detect all potentially carcinogenic HPVs and to perform reliable genotyping. The authors implemented the classic nested PCR and Sanger DNA-sequencing technology for routine HPV testing. The results showed a true negative HPV PCR invariably indicates the absence of precancerous cells in the cytology samples. However, 80.5% of single positive HPV-16 tests and 97.3% of single positive HPV-18 tests were associated with a negative or a largely self-reversible Pap cytology. Routine sensitive and reliable HPV type-specific or perhaps even variant-specific methods are needed to address the issues of persistence of HPV infection if a virology-based primary cervical screen is used to replace the Pap cytology screening paradigm.

A highly conserved 357-bp segment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.

BackgroundA sensitive and analytically specific nucleic acid amplification test (NAAT) is valuable in confirming the diagnosis of early Lyme disease at the stage of spirochetemia.FindingsVenous blood drawn from patients with clinical presentations of Lyme disease was tested for the standard 2-tier screen and Western Blot serology assay for Lyme disease, and also by a nested polymerase chain reaction (PCR) for B. burgdorferi sensu lato 16S ribosomal DNA. The PCR amplicon was sequenced for B. burgdorferi genomic DNA validation. A total of 130 patients visiting emergency room (ER) or Walk-in clinic (WALKIN), and 333 patients referred through the private physicians' offices were studied. While 5.4% of the ER/WALKIN patients showed DNA evidence of spirochetemia, none (0%) of the patients referred from private physicians' offices were DNA-positive. In contrast, while 8.4% of the patients referred from private physicians' offices were positive for the 2-tier Lyme serology assay, only 1.5% of the ER/WALKIN patients were positive for this antibody test. The 2-tier serology assay missed 85.7% of the cases of early Lyme disease with spirochetemia. The latter diagnosis was confirmed by DNA sequencing.ConclusionNested PCR followed by automated DNA sequencing is a valuable supplement to the standard 2-tier antibody assay in the diagnosis of early Lyme disease with spirochetemia. The best time to test for Lyme spirochetemia is when the patients living in the Lyme disease endemic areas develop unexplained symptoms or clinical manifestations that are consistent with Lyme disease early in the course of their illness.