PCR Positivity Rates Research Articles

PCR Diagnosis of Invasive Candidiasis: Systematic Review and Meta-Analysis

Invasive candidiasis (IC) is a significant cause of morbidity and mortality. Diagnosis relies on culture-based methods, which lack sensitivity and delay diagnosis. We conducted a systematic review assessing the diagnostic accuracy of PCR-based methods to detect Candida spp. directly in blood samples. We searched electronic databases for prospective or retrospective cohort and case-control studies. Two reviewers abstracted data independently. Meta-analysis was performed using a hierarchical logistic regression model. Random-effects metaregression was performed to assess the effects of study methods and infection characteristics on sensitivity or specificity values. We included 54 studies with 4,694 patients, 963 of whom had proven/probable or possible IC. Perfect (100%) sensitivity and specificity for PCR in whole-blood samples was observed when patients with cases had candidemia and controls were healthy people. When PCR was performed to evaluate patients with suspected invasive candidiasis, the pooled sensitivity for the diagnosis of candidemia was 0.95 (confidence interval, 0.88 to 0.98) and the pooled specificity was 0.92 (0.88 to 0.95). A specificity of >90% was maintained in several analyses considering different control groups. The use of whole-blood samples, rRNA, or P450 gene targets and a PCR detection limit of ≤ 10 CFU/ml were associated with improved test performance. PCR positivity rates among patients with proven or probable IC were 85% (78 to 91%), while blood cultures were positive for 38% (29 to 46%). We conclude that direct PCR using blood samples had good sensitivity and specificity for the diagnosis of IC and offers an attractive method for early diagnosis of specific Candida spp. Its effects on clinical outcomes should be investigated.

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

Clinical Review of Pulmonary Tuberculosis in Teenagers According to the Involved Lung

Purpose : The purpose of this study was to investigate clinical features and culture-positive rates according to the involved lung in adolescent pulmonary tuberculosis (TB). Methods : We retrospectively reviewed the medical records of adolescents who ranged in age from 10 to 20 years and who had been hospitalized with a diagnosis of TB at Kwangju Christian Hospital from 2000 to 2008. Results : Sixty-six patients were identified with pulmonary TB: median age 16.82 years; 48.5% males. Among them, 90.9% of patients were between 15 and 20 years of age. Most patients presented with multiple symptoms, and the most common included cough (74.2%), sputum (60.6%), fever (39.5%), and night sweating (18.2%). Sputum samples were smear-positive in 28 (42.4%), culture-positive in 40 (60.6%), and PCR-positive in 46 (69.7%). The most common radiological patterns included cavitation in 18 (27.3%), pleural effusion in 18 (27.3%), lymphadenopathy in 10 (15.2%), and tuberculoma in 5 (7.6%). The prevalence of smear, culture, and PCR positive rates increased as the number of involved lobes increased (P

Study of HIV seroprevalence and sociodemographic patterns in antenatal cases and vertical transmission to neonates

The number of people living with HIV has been increasing in every region. With 27 million pregnancies a year and an overall estimated 0.03% prevalence rate of HIV infection among pregnant women it is estimated that about 100000 HIV infected women deliver every year. Using a conservative vertical transmission rate of 30% about 30000 infants acquire HIV infection each year. In Maharashatra HIV seroprevalences is >1% in women attending antenatal clinics. This study conducted in a general public hospital for a period of one year from October 2001 to September 2002 aimed at screening of antenatal mothers to find HIV seroprevalence sociodemographic patterns in HIV seropositive mothers and reverse transcriptase PCR positivity in babies born of HIV positive mothers during first week of the birth. All pregnant women attending the antenatal clinics were screened for HIV sero-prevalence as per NACO guidelines. Reverse Transcriptase PCR (RT PCR) was done on neonatal serum samples collected during first five days of birth and the test done as per instruction of the manufacturer. During the study period 2550 pregnant women were screened for HIV and 1.86% (52) were found to be HIV seropositive. Of these 52 seropositive women 47 gave consent for the HIV testing of their babies which were included in further studies. The average age of these women was 23.78 +or- 3.75 years. Out of the 47 women 76.6% were residing in slums and 25.5% comprised of migratory population. Among the seropositive women 42.6% had education till secondary level while 38.3% were illiterate and 19.1% had primary education. In this study 57.4% of the spouses were unskilled worker followed by 19.1% in service 12.8% factory workers and 10.6% were drivers/cleaners. Primigravida women were 53.2% and 34% were second gravida. Of the 47 families 93% were from low socio-economic class. The mean time of collection of blood of the newborn was 2.85 days. There is no precise data available on the percentage of infants infected at birth. The RT PCR done on the neonatal sera samples was positive for 31.9% (15) babies indicating probable in utero transmission of HIV infection. Follow-up RT PCR is needed to confirm the transmission of HIV infection in these neonates. Among the women delivered by caesarean section the rate of RT PCR positivity in infants was high (10/16) than women who delivered normally (05/31). As the study group was small the relationship between the caesarean section and the rate of transmission could not be established. (full text)

The Suaeda liaotungensis kitag betaine aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with minimum linear length of DNA fragment

In this research we established a particular vector-free and marker-free plant transformation system of maize to overcome the obstacles of biosafety limits. The BADH gene was introduced into maize by pollen-tube pathway, using the principle of minimum linear length of the transformation element, which was composed of only the BADH gene, expression regulatory sequence (35S CAMV promoter, NOS terminator), and T-DNA border sequence at both sides. Twenty-seven of 2076 transformed samples were positive in PCR amplification and the PCR positive rate of T1 generation was 1.3%. Further Southern blotting results indicated that the BADH gene was integrated into maize genome. Transgenic lines of progeny were examined for tolerance to NaCl by induced salt stress with 250 mM NaCl Hoagland solution. After 15 days of treatment, 73.9–100% of the transgenic seedlings survived and grew well, whereas most wild-type seedlings wilted and showed loss of chlorophyll. Only 8.9% of the wild-type plants survived but gradually died after salt stress. The electrical conductivity of the transgenic line of progeny after salt stress was lower than wild type. The transgenic progeny had higher glycinebetaine and Chlorophyll content than wild type after salt stress.

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target

The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.

Detection of herpes simplex virus type 1 DNA in bilateral human trigeminal ganglia and optic nerves by polymerase chain reaction

Herpes simplex virus type 1 (HSV-1) DNA was extracted from human trigeminal ganglia of 121 cadavers aged between 3 months and 94 years, and its PCR amplification was performed for the RL2 HSV-1 sequence using two pairs of primers. The HSV-1 DNA was detected in 74 of 121 patients (61%); 70/74 bilaterally, 3/74 only on the left side, and 1/74 only on the right side. Although the PCR-positive rate was significantly higher in advanced age, the correlation between the PCR-positive rate and gender was unclear. Additionally, HSV-1 DNA was not detected in any of the 50 optic nerve samples.

Real-time quantitative RT-PCR detection of disseminated endometrial tumor cells in peripheral blood and lymph nodes using the LightCycler System

Objective. Some endometrial cancer patients without clinical evidence of extrauterine spread die as a result of recurrence even after curative operation. These recurrences may arise from occult tumor cells that are not detected by conventional methods. The goal of this study was to develop a quantitative method for the detection of disseminated tumor cells (DTCs) in the peripheral blood (PB) and lymph nodes (LNs) of patients with endometrial cancer. Methods. Ninety-eight PB samples from 30 patients and 218 LNs from 14 patients were studied. Real-time quantitative analysis was performed using a LightCycler instrument and a TaqMan probe for cytokeratin 19 (CK19) as a marker gene. Results. This method resulted in the reproducible quantitation of 10 to 10 6 MCF-7 cells (CK19-expressing breast cancer cell line) per 10 6 peripheral blood nucleated cells. CK19 mRNA expression was detected in 28 PB samples and in 62 LNs. Only three preoperative PB samples and one postoperative PB sample (from four patients) and 33 LNs (from six patients) were PCR-positive. The PCR-positive rate of LNs was higher in patients with pathologically metastatic (path-positive) LNs than in patients with path-negative but PCR-positive LNs. Furthermore, the CK19 mRNA background expression rate was higher in the LNs of path-negative but PCR-positive patients than in LNs of path-negative and PCR-negative patients. Conclusions. Real-time qRT-PCR with TaqMan probes is a sensitive, specific and rapid method for the detection of DTCs in PB and LNs. Additional studies with larger numbers of patients and adequate follow-up would be of benefit.

Toltrazuril treatment to control diaplacentalNeospora caninumtransmission in experimentally infected pregnant mice

We addressed the question whether diaplacental transmission of Neospora caninum can be controlled by metaphylactic chemotherapy using toltrazuril or enrofloxacin. Female C57/BL6 mice, infected on day 10 of pregnancy, were medicated for 6 consecutive days p.i. with 52.5 mg toltrazuril or - as an out-group control medication--16.7 mg enrofloxacin per kg body weight per day. Other control groups received either infection but no medication or vice versa. Toltrazuril treatment significantly reduced pre- and perinatal losses (10 deliveries of healthy newborns, versus 1 abortion and 4 failures) when compared to control-enrofloxacin (2 deliveries, versus 1 abortion, 7 failures and 2 pre-parturient deaths of dams) and non-treated animals (3 deliveries, versus 6 abortions, 8 failures and 4 pre-parturient deaths). Simultaneously, PCR-based parasite detection in the brain of mothers, histopathological findings as well as clinical fatality were significantly less frequent in toltrazuril-treated dams. The overall toltrazuril treatment efficacy was determined as 87 %, that of enrofloxacin-treatment as 17 %. The progenies of toltrazuril-treated dams also exhibited a very low rate of PCR-positivity in their brain (3 out of 39), whereas untreated dams delivered litters with mostly PCR-positive brains (12 out of 14) and a relatively high death rate post-partum (5 out of 19 newborns died). Mice subjected to a second mating delivered newborns all negative by N. caninum-PCR, indicating that diaplacental tachyzoite passage does not occur in a later, repeated pregnancy. Overall, our experiments showed that toltrazuril-treatment of an acute N. caninum-infection--induced during pregnancy--results in a clear reduction of fetal losses and a marked reduction of diaplacental passage of the parasite to the fetal brain, whereas enrofloxacin, as an out-group control substance, failed to show the same effect.

P0293 FINAL EVALUATION OF A LOOKBACK PROCEDURE TO IDENTIFY HCV INFECTION IN THE TRANSFUSED NICU POPULATION

Introduction: Transfusions of blood and blood products were frequently administered to infants admitted to the NICU nursery in the period between 1978–1989 before the identification of the Hepatitis C virus. This lookback study encompasses three distinct time periods between Jan 1978 and Oct 1985 (pre-HIV screening), Nov 1985–Apr 1990 (pre 1st generation HCV testing) and May 1990–Mar 1992 (pre 2nd generation HCV testing) –Periods 1,2 and 3 respectively. Specific aims were to: 1. Determine the incidence and natural history of HCV infection in infants transfused between Jan 1978 and Mar1992. 2. Assess parental awareness of transfusion 3. Examine the efficacy and feasibility of a lookback procedure using various methods of contact (medical records address, provincial health insurance database, provincial archival look-back, provincial laboratory database) Methods: Records from 21,248 (96.9%) of the 21,928 admissions to the two Regional NICU’s in Edmonton were reviewed for transfusions. A package including a requisition for HCV Ab and PCR testing and a questionnaire regarding knowledge of transfusion and its associated risks was mailed to identified transfused infants’ caregivers using the methods of contact above. Results: 7590 (36%) of the infants were transfused. Results are now expressed according to Periods 1, 2 and 3. Transfused neonates decreased over time from 42.4% to 29.9% to 19.4%. The transfused group had significantly higher in-hospital mortality proportions than the non-transfused group (14.4%, 16.4%, 17.2% vs 3.6%, 2.5%, 5%). Awareness of transfusion was 46.3%, 51.2% and 60.1%. Successful testing responses were 31.5%, 48.8% and 45.2%. Of children tested for HCV Ab, 17 (1.5%), 12 (1.5%) and 1 (0.4%) children were positive and 3 subjects in each of periods 1 & 2 were HCV-indeterminate. The rate of Hepatitis C PCR positivity in the whole group positive for HCV antibody was 67%. Crude per donor risks of HCV in the three periods were 0.0027, 0.0028 and 0.0005 respectively. Conclusion: Transfusions decreased over time in both NICU’s, but transfused infants had significantly higher in-hospital mortality than non-transfused infants. Parental awareness of transfusion increased over time, as did successful testing for HCV Ab. The rate of HCV infection in the NICU’s was 1.5% for the period prior to the introduction of 1st generation HCV Ab testing, and decreased thereafter, as did crude per donor HCV risk. The rate of PCR positivity in our group of HCV positive patients was 67%. We acknowledge the support of CIHR, MRC, Canadian Blood Services, the Canadian Red Cross Society and the Children’s Health Foundation of Northern Alberta.

Detection rates of bacteria in chronic otitis media with effusion in children.

This study was performed to investigate polymerase chain reaction-based detection of bacterial DNA in middle ear fluid and assess the correlation between the PCR-positive rate with several factors associated with middle ear effusion. The purpose was to gain a further understanding of bacterial infection as a major cause of otitis media with effusion. Of the 278 specimens of middle ear fluid, 39 (14%) tested positive by ordinary culture. The overall detection rate of bacterial DNA using the PCR method was 36.7% for middle ear effusion, and bacterial DNA detection rates of Hemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis in the middle ear effusion were 29.1%, 4.7% and 10.8%, respectively. The bacterial DNA detection rate was higher in ears with a history of acute otitis media than those without the history. High detection rates were observed in patients younger than 48 months who have had a higher tendency to present with acute otitis media. We concluded that PCR is a more sensitive method for the detection of bacteria in middle ear effusion than ordinary culture, and acute otitis media is a major contributor to the pathogenesis of otitis media with effusion.

Molecular detection and characterization of circulating tumor cells and micrometastases in prostatic, urothelial, and renal cell carcinomas.

The detection and molecular characterization of circulating tumor cells (CTCs) and micrometastases in urinary tract and prostatic tumors may have important prognostic and therapeutic implications. In the last decade, numerous groups have attempted the detection of occult tumor cells in renal, prostatic, and urothelial carcinomas using the highly sensitive reverse-transcriptase polymerase chain reaction (RT-PCR). In prostatic carcinoma (PC), tissue-specific transcripts were detected with high specificity in the blood of patients with localized and advanced disease. PCR assays for PC detection were shown to be strong predictors of poorer outcome in some reports, while a lack of prognostic significance was found in other studies. There was a vast difference in the PCR positivity rates between various groups studying PC. This discrepancy could be due to variations in PCR methodology. In urothelial and renal cell carcinoma, the amount of research on the subject is still too limited. Currently, these assays for occult tumor cells are promising but are not yet ready to use in PC and urinary tract tumors. Because of the many limitations of PCR (e.g., false positives), many groups are developing new approaches for the detection of occult tumor cells. The most attractive technique involves immunomagnetic isolation of intact CTC and micrometastases prior to downstream analysis. The tumor-rich magnetic fraction can be subjected to RT-PCR, immunocytochemistry, and in situ hybridization. This will lead to better quantification and molecular characterization of these tumor cells.

Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned?

The detection of circulating tumor cells (CTC) and bone marrow micrometastases (BMM) by reverse transcriptase polymerase chain reaction (RT-PCR) may help predict recurrence and survival in malignant melanoma (MM). Since the appearance of the original article by Smith et al. in 1991 (Lancet 338:1227), several groups have attempted the detection of CTC and BMM in MM using RT-PCR for melanocytic specific markers, mainly tyrosinase mRNA. Most studies show that tyrosinase is not present in the PB and BM of control individuals without MM. The PCR positivity rates in MM are extremely variable, ranging from 0% to 100%. There was a correlation between RT-PCR and stage in some but not all of the studies. These disparate findings could in part be explained by differences in RNA extraction and blood separation techniques, to unrecognized contamination leading to false positive results, or differences in patient selection. Despite these discrepancies, we and others have shown that RT-PCR for tyrosinase mRNA in PB is able to predict overall survival (OS) and disease-free survival (DFS) in a statistically significant manner. In AJCC stage II-IV patients rendered surgically free of disease, we found that blood tyrosinase positivity was an independent predictor of OS and DFS. We also found that BM tyrosinase positivity is an independent predictor of DFS in the same group of patients. RT-PCR may help identify subgroups of patients at high risk for early relapse for more aggressive adjuvant therapy. Large prospective studies and interlaboratory quality assurance initiatives are necessary to confirm the accuracy and prognostic value of these RT-PCR assays.

ChemInform Abstract: Molecular Detection of Micrometastases and Circulating Tumor Cells in Melanoma Prostatic and Breast Carcinomas

The molecular detection of circulating tumor cells (CTC) and micrometastases may help develop new prognostic markers in patients with solid tumors. In the last 10 years, numerous groups have attempted the detection of occult tumor cells in solid malignancies using the highly sensitive reverse transcriptase polymerase chain reaction (RT PCR) technique. These assays were in the vast majority directed against tissue specific markers. In most studies on prostatic carcinoma, RT PCR was able to specifically detect prostatic tissue specific markers in the peripheral blood (PB), bone marrow (BM) and lymph nodes of patients with localized and metastatic disease. Melanoma related transcripts were detected by RT PCR in the PB, BM and lymph nodes of patients with localized and advanced tumors. In most studies, melanoma related markers were shown to be specific except when assayed in lymph nodes. RT PCR positivity rates were highly variable between studies. Despite tFlese discrepancies, many authors have shown a statistically significant correlation between RT PCR positivity and a poorer outcome in both melanoma and prostatic carcinoma. In breast carcinoma, all markers that have been extensively tested were shown to be non-specific. Because of the many limitations of RT PCR (e.g. false positives), many groups are developing new approaches for the detection occult tumor cells. One of these techniques involves immunobead isolation of CTC and micrometastases prior to down stream analysis. The tumor rich magnetic fraction can be subjected to RT PCR, immunocytochemistry and flow cytometry. In conclusion, the molecular detection of occult tumor cells in solid tumors seems very promising and the techniques used for this purpose are in continuous evolution. Large prospective and interlaboratory variability studies are necessary to determine the accuracy and prognostic value of these assays.

Improved methods of detection of meningococcal DNA from oropharyngeal swabs from cases and contacts of meningococcal disease.

In the UK the increasing use of pre-admission parenteral antibiotic therapy in meningococcal disease has lessened the value of routine cultures as a tool to confirm diagnosis, and laboratory confirmation of invasive meningococcal infection is achieved increasingly by non-culture, nucleic acid amplification methods. The purpose of this study was to evaluate a DNA extraction and meningococcal-specific DNA amplification methodology for detection of meningococci from oropharyngeal swabs. One hundred and six swabs from suspected or confirmed cases of meningococcal disease, and 94 swabs from contacts of meningococcal disease cases were examined. Of laboratory-confirmed cases, 38/65 (58.5%) yielded a positive oropharyngeal swab PCR result and 5/24 (20.8%) swabs from suspected but laboratory-unconfirmed cases were PCR positive. No significant differences in PCR positivity rates were found between the types of swab transport systems utilized, but transport time to the testing laboratory was found to affect PCR positivity (P < 0.05). Application of meningococcus-specific PCR to oropharyngeal swabs, in addition to routine culture of swabs, can provide valuable epidemiological information as well as case confirmation for contact management. PCR amplification of meningococcal PCR from oropharyngeal swabs will also increase the ascertainment in swabbing surveys carried out as part of meningococcal disease outbreak investigation and management.

Clinical use of capillary PCR to diagnose Mycoplasma pneumonia.

In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of Mycoplasma infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with Mycoplasma pneumonia. We performed PCR analysis for Mycoplasma DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom Mycoplasma pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum Mycoplasma antibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing Mycoplasma pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results.

Detection of circulating tumor cells in patients with prostate cancer using prostate specific membrane-derived primers in the polymerase chain reaction.

Prostate specific membrane antigen (PSM) is a transmembrane glycoprotein that has been described as human prostate specific. It is possible that PSM could be used as a biomarker for staging prostate cancer. Peripheral blood mononuclear cells from 2 groups of patients with prostate cancer were used for the detection of PSM messenger ribonucleic acid (mRNA) using the reverse transcriptase-polymerase chain reaction (RT PCR) method. Group 1 consisted of 29 untreated patients (13 stage B cancer, 5 stage C, and 11 stage D cancers). Group 2 consisted of 40 treated patients (23 responded well and 17 had recurrence after treatment). In addition, blood specimens from 30 patients with benign prostatic hyperplasia, 5 women, and 5 men undergoing cystoprostatectomy for invasive bladder cancer were used as controls. We then correlated patient and disease characteristics with PCR assay results. Samples of all 40 controls were negative for PSM-mRNA. Thirteen of 29 patients of group 1 (45%) were positive for PSM-mRNA. The PCR positive rate did not correlate with clinical stage, pathologic stage, tumor grade, or serum PSA levels. Nine of 40 patients (22%) in group 2 were positive for PSM-mRNA, and the majority (8 of 9) were derived from the group of patients with recurrent disease. A nested RT PCR assay for PSM mRNA can detect circulating tumor cells in the peripheral blood of patients with prostatic cancer. These results suggest that the detection of circulating tumor cells could be useful for monitoring disease progression of prostatic cancer.

Bloodstream invasion in early Lyme disease: Results from a prospective, controlled, blinded study using the polymerase chain reaction

The purposes of this study were to determine (1) the optimal techniques for and potential diagnostic usefulness of the polymerase chain reaction (PCR) in early Lyme disease, and (2) the true frequency and clinical correlates of PCR-documented blood-borne infection in the dissemination of Lyme disease. We performed a prospective, controlled, blinded study of PCR, culture, and serology on fractionated blood samples from 105 patients; 76 with physician-diagnosed erythema migrans and 29 controls. Clinical characteristics of the patients were obtained with a standardized data entry form and correlated with results of the laboratory studies. Only 4 of the 76 (5.3%) patients with erythema migrans were culture positive; however, 14 of 76 (18.4%) had spirochetemia documented by PCR of their plasma. None of 29 controls were PCR or culture positive (P = 0.007, versus patients). PCR-documented spirochetemia correlated with clinical evidence of disseminated disease; 10 of 33 patients (30.3%) with systemic symptom(s) were PCR positive compared to 4 of 43 (9.3%) without such evidence (P = 0.02). PCR positivity was more frequent among patients with each of four specific symptoms: fever, arthralgia, myalgia, and headache (all P < 0.05). A higher total number of symptoms (median 2.5 in PCR-positive patients versus 0 in PCR-negative controls; P < 0.01) and the presence of multiple skin lesions (37.5% of patients with multiple, versus 13.3% of patients with single lesions [P = 0.04] were also correlated with PCR positivity. Patients with both systemic symptoms and multiple skin lesions had a 40% PCR-positivity rate; however, 4 of 42 (9.5%) asympatomatic patients with only single erythema migrans lesions were also PCR positive. In multivariate analysis using logistic regression, the number of systemic symptoms was the strongest independent predictor of PCR positivity (P = 0.004). PCR detection of Borrelia burgdorferi is at least three times more sensitive than culture for identifying spirochetemia in early Lyme disease and may be useful in rapid diagnosis. PCR positivity significantly correlates with clinical evidence of disease dissemination. Bloodstream invasion is an important and common mechanism for the dissemination of the Lyme disease spirochete.

Detection of Epstein-Barr virus and cytomegalovirus genome in white blood cells from patients with juvenile rheumatoid arthritis and childhood systemic lupus erythematosus.

The role of infectious agents in the pathogenesis of autoimmune diseases has long been a matter of debate. This study investigated the possible role of Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) infections in the pathogenesis of autoimmune diseases by an attempt to demonstrate the presence of the viral genome in the leukocyte of 21 juvenile rheumatoid arthritis (JRA) patients, 20 childhood-onset systemic lupus erythematosus (SLE) patients, and 20 age-matched normals, using polymerase chain reaction (PCR) and DNA probes. The results showed: (1) there was no difference in serum IgG anti-EBV antibody titers among three groups; (2) the EBV PCR-positive rates for JRA and SLE patients and normal controls were 5% (1/21), 10 (2/20), and 0% (0/20), respectively; (3) the HCMV PCR-positive rates for JRA and SLE patients and normal controls were 33% (7/21), 25 (5/20), and 10% (2/20), respectively, and (4) the HCMV-positive rate was 25% for JRA patients with steroid treatment and 33% for those without steroid treatment. It is, therefore, concluded that: (1) the data do not support the participation of EBV and HCMV in the pathogenesis of childhood-onset SLE and JRA; (2) steroid therapy does not increase the frequency of HCMV infection in JRA patients, and (3) immunoincompetence might be one of the major factors contributing to increased susceptibility to HCMV infection in JRA and SLE patients.

非放射性プローブを用いたPolymerase Chain Reaction法によるHuman Immunodeficiency Virus Type-1 (HIV-1) DNAの検出とウイルス分離との比較検討

We compared the results obtained with the polymerase chain reaction (PCR) and virus isolation from peripheral blood mononuclear cells (PBMC) in HIV seropositive and seronegative persons. Three primer pairs of SK38/39 (gag). SK29/30 (LTR) and SK68/69 (env) were used in the amplification of the HIV DNA sequences, and KM29/38 (beta-globin) was used as the inner control. The PCR-positive rate among the virus-isolation-positive persons was SK38/39:100% (22/22), SK29/30:95.5% (21/22) and SK68/69:90.0% (20/22). The PCR-positive rate among the virus-isolation-negative persons was SK38/39:60% (6/10), SK29/30:60% (6/10) and SK68/69:80% (8/10), and two subjects were PCR-negative with all primer pairs. We could not detect HIV DNA from seronegative samples, and all subjects were positive with the inner control. Each primer pair expressed a different PCR-positive rate. There are possible explanations for the low PCR-negative rate on virus-isolation negative-subjects that the number of infected cell was rare or infected HIV contained genetic variations or deletions. We considered that the results of PCR correlated with the character of HIV as infectivity.