Automated Polymerase Chain Reaction Research Articles

Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens.

The rise of highly transmissible SARS‐CoV‐2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS‐CoV‐2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS‐CoV‐2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.

Feasibility and performance of the Idylla™ NRAS/BRAF cartridge mutation assay on thyroid liquid-based fine-needle aspiration.

Thyroid nodules with indeterminate cytology represent up to 30% of cases. Molecular testing is now highly recommended to improve management. This study aimed to evaluate the use of the Idylla™ NRAS/BRAF mutation test, a rapid and automated polymerase chain reaction (PCR) assay validated for fixed paraffin-embedded use, on residual thyroid liquid-based fine-needle aspiration (LB-FNA). Concordance between mutations detected by the Idylla™ assay and the gold-standard qPCR was assessed by splitting in two aliquots 31 BRAF or RAS mutated and 5 non-mutated LB-FNA samples. Samples were obtained either from simulated FNA after thyroidectomy or from FNA obtained during routine care. A third aliquot was used to assess the limit of detection of Idylla™ for five mutated samples. The Idylla™ assay showed a sensitivity of 97% and a specificity of 83% as results were concordant for 34 out of 36 samples. One discordant sample concerned a BRAF p.K601E-mutation which is not detected by the Idylla™ cartridge. The other showed a false-positive NRAS p.A146T detection and a weak BRAF p.V600E detection. The limit of detection of the Idylla™ assay was not reached by the dilution assay. Idylla™ NRAS/BRAF mutation testing can be performed on residual thyroid LB-FNA, using low DNA quantity input, thus not requiring a dedicated sample. The Idylla™ NRAS/BRAF assay offers a quick and easy first step for analyzing the main molecular alterations in indeterminate thyroid nodules, hence improving diagnostic management.

Performance Comparison of a Novel Rapid Stand-alone Molecular Test and a 2-Step Diagnostic Algorithm for Clostridioides difficile Detection in Children.

We aimed to evaluate diagnostic performance of the cobas® Liat® Cdiff test, a novel single-step automated polymerase chain reaction (PCR) assay for rapid diagnosis of toxigenic Clostridioides difficile infection (CDI) in stool samples from children with clinical symptoms of CDI. Assessment of cobas Liat Cdiff diagnostic yield, time of analytical process and agreement of results with those of a 2-step diagnostic algorithm. The sequential algorithm combined an enzyme immunoassay (EIA) targeting antigen glutamate dehydrogenase (GDH), enterotoxin-A and cytotoxin-B, and a confirmatory PCR in EIA GDH-positive and toxin-negative samples. Fresh stool samples were collected prospectively from patients 2-18 years of age that were attended in Hospital Sant Joan de Deu (Barcelona, Spain) during December 2018-August 2019. A total of 122 specimens were collected from 91 children (mean age, 8 years; 69.7% male). cobas Liat Cdiff identified 24 (19.7%) positive samples. EIA yielded 97 (79.5%) GDH- and toxin-negative results, 11 (9.0%) GDH- and toxin-positive results, and 14 (11.5%) GDH-positive and toxin-negative results, of which 11 (9.0%) were positive for the toxin by the confirmatory PCR. Overall, GDH- and toxin-positive samples detected by the sequential algorithm were 22 (18.0%). Comparatively, the new test reduced time of the analytical process significantly (20 vs. 35.4 minutes, P < 0.001). Use of cobas Liat Cdiff showed similar detection yield compared with a 2-step diagnostic algorithm that combined an EIA and a confirmatory PCR while decreasing the time of the analytical process markedly in stool samples from children suspected of CDI.

Cerebrospinal Fluid Bacillary Load by Xpert MTB/RIF Ultra Polymerase Chain Reaction Cycle Threshold Value Predicts 2-Week Mortality in Human Immunodeficiency Virus-Associated Tuberculous Meningitis.

BackgroundThe World Health Organization recommends GeneXpert MTB/RIF Ultra (Xpert Ultra), a fully automated polymerase chain reaction (PCR) assay, as the initial tuberculous meningitis (TBM) diagnostic test. The assay’s PCR cycle threshold (Ct) values represent the number of PCR cycles required for probe signal to be detected (low Ct value = high bacillary load) and may approximate tuberculosis (TB) bacillary load. We measured the relationship between cerebrospinal fluid (CSF) TB bacillary load with mortality. MethodsWe prospectively enrolled 102 human immunodeficiency virus (HIV)–positive Ugandans with probable or definite TBM from April 2015 to August 2019. Xpert Ultra Ct tertiles and semi-quantitative categories were separately analyzed as predictors of 2-week mortality. We investigated associations between Ct and baseline clinical and CSF parameters. ResultsSubjects with Ct values in the low tertile (ie, high bacillary load) had 57% 2-week mortality—worse than the intermediate (17%) and high (25%) Ct tertiles and Xpert Ultra–negative (30%) probable TBM cases (P = .01). In contrast, the reported semi-quantitative Xpert Ultra categorization was less precise; with the medium to low category trending toward worse 2-week survival (42%) compared with very low (28%), trace (26%), and negative (30%) categories (P = .48). Ct tertile was significantly associated with baseline CSF lactate (P = .03). ConclusionsHigh CSF TB bacillary load, as measured by Xpert Ultra Ct tertile, is associated with an almost 2-fold higher 2-week mortality in HIV-associated TBM and is a better predictor than the reported Xpert Ultra semi-quantitative category. Xpert Ultra Ct values could identify TBM patients at increased risk of death who may benefit from enhanced supportive care.

Development of a DNA isolation device using poly(3,4-dihydroxy-L-phenylalanine)-coated swab for on-site molecular diagnostics

For on-site molecular diagnostics, a pre-treatment step for isolation of nucleic acid from clinical samples on site is desired. However, conventional commercialized silica-based nucleic acid isolation kits require repetitive pipetting and a centrifugation or permanent magnet for buffer exchange. In this study, we developed a poly(3,4-dihydroxy-L-phenylalanine) (L-DOPA)-coated swab that can absorb and desorb DNA depending on pH of buffers and a portable integrated DNA isolation device that comprises integrated chambers containing DNA isolation buffers. The poly(L-DOPA)-coated swab interacts with each buffer by passing through the membrane between the integrated chambers. Our device involves a simple operation and does not require any large equipment or skilled experts. By connecting the device with an automated polymerase chain reaction system, an isothermal amplification system, or a non-amplified DNA detection method, on-site molecular diagnosis of various diseases can be quickly realized.

Value of PCR in sonication fluid for the diagnosis of orthopedic hardware-associated infections: Has the molecular era arrived?

IntroductionBone healing disturbance following fracture fixation represents a continuing challenge. We evaluated a novel fully automated polymerase chain reaction (PCR) assay using sonication fluid from retrieved orthopedic hardware to diagnose infection. Patients and methodsIn this prospective diagnostic cohort study, explanted orthopedic hardware materials from consecutive patients were investigated by sonication and the resulting sonication fluid was analyzed by culture (standard procedure) and multiplex PCR (investigational procedure). Hardware-associated infection was defined as visible purulence, presence of a sinus tract, implant on view, inflammation in peri-implant tissue or positive culture. McNemar’s chi-squared test was used to compare the performance of diagnostic tests. For the clinical performance all pathogens were considered, whereas for analytical performance only microorganisms were considered for which primers are included in the PCR assay. ResultsAmong 51 patients, hardware-associated infection was diagnosed in 38 cases (75%) and non-infectious causes in 13 patients (25%). The sensitivity for diagnosing infection was 66% for peri-implant tissue culture, 84% for sonication fluid culture, 71% (clinical performance) and 77% (analytical performance) for sonication fluid PCR, the specificity of all tests was >90%. The analytical sensitivity of PCR was higher for gram-negative bacilli (100%), coagulase-negative staphylococci (89%) and Staphylococcus aureus (75%) than for Cutibacterium (formerly Propionibacterium) acnes (57%), enterococci (50%) and Candida spp. (25%). ConclusionThe performance of sonication fluid PCR for diagnosis of orthopedic hardware-associated infection was comparable to culture tests. The additional advantage of PCR was short processing time (<5 h) and fully automated procedure. With further improvement of the performance, PCR has the potential to complement conventional cultures.

Molecular detection of Salmonella species in bovine fecal samples

A study was performed to assess the validity of the BAX automated polymerase chain reaction system (DuPont Nutrition & Health, Wilmington, Delaware) to detect the shedding of Salmonella species in bovine fecal samples. A total of 133 bovine fecal samples that were submitted to the Cornell University Animal Health Diagnostic Laboratory for Salmonella culture were also tested in the BAX system with a modified version of the manufacturer's enrichment protocol. Using culture as the gold standard test, the BAX system was found to have a sensitivity of 85.7% and a specificity of 90.5%. There was excellent agreement (κ = 0.71, standard error = 0.072) and no significant differences between the 2 methods (McNemar χ(2) = 0.180).

Xpert MTB/RIF®, a novel automated polymerase chain reaction–based tool for the diagnosis of tuberculosis [Technical note

There is an urgent need for new point of care tests for tuberculosis (TB). Xpert MTB/RIF® is a real-time polymerase chain reaction-based system that detects Mycobacterium tuberculosis DNA and rifampicin (RMP) resistance modulating mutations directly from clinical samples in 2 h. The sensitivity for detecting M. tuberculosis in culture-positive samples was 93.8% (60/64) and exceeded smear microscopy (40/64, 62.5%). The specificity for detecting M. tuberculosis was 92.0% (23/25) and for RMP resistance it was 100% (8/8). The test is simple to conduct and requires basic sputum handling facilities only. These characteristics render it a promising close-to-patient test for TB in various settings.

Prospective, Multicenter Evaluation of the BD GeneOhm VanR Assay for Direct, Rapid Detection of Vancomycin-Resistant Enterococcus Species in Perianal and Rectal Specimens

The BD GeneOhm VanR assay (BD Diagnostics, San Diego, CA), a qualitative test for the rapid detection of vancomycin-resistant enterococci (VRE) from rectal and/or perianal swabs, combines integrated nucleic acid extraction and automated polymerase chain reaction for the detection of vanA and/or vanB gene sequences. We studied 1,027 perianal and rectal swab specimens from 3 geographically distinct US sites (prevalence rates, 13.1%-25.8%). Direct swab specimens were tested by the assay and compared with direct culture. The sensitivity, specificity, and positive and negative predictive values of the assay were 93.2%, 81.9%, 54.4%, and 98.1%, respectively. The specificity was limited largely due to false-positives in the vanB portion of the assay. Specificity with perianal swabs was significantly greater than with rectal swabs, 87.1% vs 74.7%, respectively (P < .0001). When used only to detect resistance conferred by vanA, the assay was 88.3% (158/179) sensitive and 95.8% (802/837) specific, with positive and negative predictive values of 81.9% and 97.4%, respectively. The assay is a simple, rapid, and acceptable method for screening for VRE in a variety of populations in which vanA is the predominant genotype. Samples positive for the vanB genotype should be confirmed by culture owing to the apparent high number of false-positive results.

Comparison of Polymerase Chain Reaction of Polymorphonuclear Leukocytes and Plasma Identifies Patients Who Control Cytomegalovirus Infection after Hematopoietic Cell Transplantation

By use of an automated polymerase chain reaction test of plasma and a qualitative polymerase chain reaction assay on polymorphonuclear leukocytes, we identified a subgroup of hematopoietic cell transplant recipients who were able to control cytomegalovirus infection early after hematopoietic cell transplantation without antiviral therapy. Thirty-one percent of patients had cytomegalovirus DNA detected by qualitative polymerase chain reaction assay but had no cytomegalovirus DNA detected by the automated test; this group maintained a lower peak cytomegalovirus load, compared with the group of patients who had cytomegalovirus DNA detected by both tests (P = .03), suggesting a greater degree of functional immune reconstitution.

Automation of DNA marker analysis for molecular breeding in crops: practical experience of a plant breeding company

AbstractIn modern plant breeding, DNA marker analyses are of increasing importance and, as the methods become more widely adopted, the capacity for high‐throughput analyses at low cost is crucial for its practical use. Automation of the analysis processes is a way to meet these requirements. In order to achieve this, while keeping adequate flexibility in the analysis processes, Svalöf Weibull AB (SW) has developed a fully automated polymerase chain reaction system. It has been evaluated on barley and canola lines and is capable of analysing up to 2200 samples per day at a cost of 0,24 € per analysis for marker‐assisted selection and quality control of genetically modified organisms. A detailed description of this system is given, and improvements to the throughput and applications are discussed.

Real-time automated polymerase chain reaction (PCR) to detect Candida albicans and Aspergillus fumigatus DNA in whole blood from high-risk patients

We report the development and evaluation of a real-time PCR assay using the LightCycler instrument for the detection of C. albicans and A. fumigatus DNA in whole blood. Recently published consensus criteria for the diagnosis of invasive fungal infection (IFI) were used for all patient samples. Unique and published primer pairs were developed and assessed for sensitivity, specificity, and reproducibility to detect C. albicans and A. fumigatus DNA in samples spiked with purified DNA, and whole blood samples from 8 high-risk patients and 45 negative controls. The real-time assay demonstrated an analytical sensitivity of 10 fg of purified C. albicans and A. fumigatus DNA and was found to be specific for each species. The standardized approach was highly reproducible and detected C. albicans and A. fumigatus DNA in two patients with proven IFI and in one patient with a possible IFI. In addition, we report for the first time the use of recently published international consensus criteria for the diagnosis of IFI in the evaluation of a mildy invasive fungal diagnostic assay. Standardized clinical criteria and a more standardized approach to detect fungal DNA in less invasive patient samples, may permit a more reliable comparison of future studies. A rapid real-time detection of fungal DNA in whole blood, combined with standard clinical markers of response, may be more useful for monitoring patients at risk of developing IFI than other diagnostic methods currently available.

A high-resolution allelotype of B-cell chronic lymphocytic leukemia (B-CLL)

The most frequent chromosomal aberrations in B-cell chronic lymphocytic leukemia (B-CLL) are deletions on 13q, 11q, and 17p, and trisomy 12, all of which are of prognostic significance. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) are used for their detection, but cytogenetic analysis is hampered by the low mitotic index of B-CLL cells, and FISH depends on accurate information about candidate regions. We used a set of 400 highly informative microsatellite markers covering all chromosomal arms (allelotyping) and automated polymerase chain reaction (PCR) protocols to screen 46 patients with typical B-CLL for chromosomal aberrations. For validation, we compared data with our conventional karyotype results and fine mapping with conventional single-site PCR. All clonal cytogenetic abnormalities potentially detectable by our microsatellite PCR (eg, del13q14 and trisomy 12) were picked up. Allelotyping revealed additional complex aberrations in patients with both normal and abnormal B-CLL karyotypes. Aberrations detectable in the samples with our microsatellite panel were found on almost all chromosomal arms. We detected new aberrant loci in typical B-CLL, such as allelic losses on 1q, 9q, and 22q in up to 25% of our patients, and allelic imbalances mirroring chromosomal duplications, amplifications, or aneuploidies on 2q, 10p, and 22q in up to 27% of our patients. We conclude that allelotyping with our battery of informative microsatellites is suitable for molecular screening of B-CLL. The technique is well suited for analyses in clinical trials, it provides a comprehensive view of genetic alterations, and it may identify new loci with candidate genes relevant in the molecular biology of B-CLL.

Performance characteristics of an automated PCR assay for the quantification of cytomegalovirus DNA in plasma

The COBAS Amplicor CMV Monitor test (Roche Diagnostics), an automated polymerase chain reaction (PCR) assay for the quantification of cytomegalovirus (CMV) DNA in plasma samples, was evaluated in a routine diagnostic laboratory. Using cell culture-derived CMV and CMV-negative human plasma, the linear detection range of the assay as well as its intra-and inter-assay variabilities were assessed. The study design allowed distinguishing variations in results related to amplification and detection from those caused by differences in the efficiency of DNA extraction. The assay was able to identify the majority of samples correctly as positive with CMV DNA concentrations above the limit of detection. However, the reported values were often twofold or more different from the (theoretical) input, which could be explained partly by inefficient DNA extraction. The following values were computed for the coefficients of determination R 2: inter-assay variability excluding DNA extraction, R 2=0.982; including DNA extraction, R 2=0.977; intra-assay variability excluding DNA extraction, R 2=0.992; including DNA extraction, R 2=0.992. On balance, the test has acceptable within-run and between-run reproducibility. It therefore allows the comparison of results obtained at different time-points as well as in different laboratories, e.g. in multi-centre studies.

Use of real-time PCR on blood samples for diagnosis of invasive aspergillosis.

We developed a new quantitative system for diagnosis of invasive pulmonary aspergillosis (IPA) using real-time automated polymerase chain reaction (PCR). Intra-assay and interassay precision rates for in vitro examination were 2.53% and 2.20%, respectively, and the linearity of this assay was obtained when there were >20 copies/well. We examined 323 samples taken from 122 patients with hematological malignancies, including 33 patients with IPA and 89 control patients. Blood samples were subjected to PCR antigen detection methods, using enzyme-linked immunosorbent assay (ELISA) and determination of plasma (1-->3)-beta-D-glucan (BDG) concentration. The sensitivities of PCR, ELISA, and BDG measurement for diagnosis of IPA were 79%, 58%, and 67%, respectively; the specificities were 92%, 97%, and 84%. Positive findings on PCR preceded those of computed tomography by -0.3+/-6.6 days, those of BDG measurement by 6.5+/-4.9 days, and those of ELISA by 2.8+/-4.1 days. Real-time PCR was sensitive for IPA diagnosis, and quantitation was accurate.

Current approaches to diagnose invasive aspergillosis: application of real-time automated polymerase chain reaction

Blood and radiological tests are used to diagnose invasive aspergillosis (IA), but it remains unknown which is more useful. We recently made a prospective study to compare the usefulness of chest CT scan, latex agglutination test (LA) and determination of plasma (1-3)-beta-D-glucan (BDG) levels for IA diagnosis. Of 215 patients, 16 were diagnosed as definite IA. While sensitivities of LA and BDG were 44% and 63%, respectively, all the patients showed some abnormal signs on chest CT scans. On average, CT scans preceded LA by 7.1 days and BDG by 11.5 days. Interestingly, false-positive rate of the blood tests increased during neutropenia. Because lung is the usual portal of Aspergillus, chest CT scan may be more beneficial than the blood tests for early diagnosis of IA. Care is necessary in interpreting the results of these tests, especially when patients are neutropenic. Blood tests must be sensitive and their results must be interpreted quantitatively. We recently developed a new quantitative diagnostic system for IA using real-time PCR. In vitro examination using 10(2) copies of Aspergillus DNA showed that linearity was obtained when there were more than 20 copies. We examined 323 samples taken from 122 patients with hematological malignancies. Blood samples were subjected to real-time PCR, enzyme-linked immunosorbent assay (EIA) and BDG. Sensitivity of real-time PCR, EIA and BDG was 79, 58 and 67%, respectively. Specificity of each assay was 92, 97 and 84%, respectively. Real-time PCR was highly sensitive for IA diagnosis, and quantification was accurate.

Quantitative proviral DNA and antibody levels in the natural history of HTLV-I infection.

The pathogenesis of human T-cell lymphotropic virus type I (HTLV-I) in adult T-cell leukemia/lymphoma (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) is poorly understood. We prospectively followed up and evaluated the virologic correlates of infection in transfusion recipients after seroconversion, in asymptomatic carriers, and in ATL and HAM/TSP patients. Proviral DNA levels (copies/105 lymphocytes) were determined by real-time automated polymerase chain reaction and antibody titers by end-point dilution by use of an HTLV-I enzyme-linked immunoassay. In early infection, proviral load was initially elevated (median, 212 copies/105 lymphocytes at time 1) and later decreased (median, 99 copies at time 2, and 27 copies at time 3). Corresponding antibody titers were low at time 1 (1:2154), had significantly increased by time 2 (1:12312), and were stable by time 3 (1:4694). These viral markers were significantly lower in asymptomatic carriers than in HAM/TSP or ATL patients. Therefore, proviral load and antibody titers may be useful as predictive markers of disease among carriers.

Detection of male genital infection with Chlamydia trachom atis and Neisseria gonorrhoeae using an automated multiplex PCR system (Cobas AmplicorTM)

Summary: We evaluated Cobas AmplicorTM, a highly automated polymerase chain reaction (PCR) system, to test first-void urine (FVU) and urethral swab specimens for Chlamydia trachomatis and Neisseria gonorrhoeae in men attending a sexually transmitted infection (STI) clinic. Results were compared against an inhouse radioimmune dot blot (DB) test for C. trachomatis and selective culture for N. gonorrhoeae . Three hundred and ninety sets of specimens were obtained from 378 consecutive new and returned-new patients. Gonorrhoea prevalence was 9.49%, with no significant difference in sensitivity or specificity between culture and PCR. Chlamydia prevalence was 15.4%, with sensitivities of: DB 55%, PCR of FVU 86.7%, urethral swab PCR 90%. The specificity of PCR on FVU and urethral swabs was 100%. We have shown that Cobas AmplicorTM PCR is highly sensitive and specific in the diagnosis of chlamydia and gonorrhoea in men attending an STI clinic. Further economic and scientific studies are needed to determine the costeffectiveness of this technique for screening in primary care settings.

Automated polymerase chain reaction product sample preparation for capillary electrophoresis analysis.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.

Quantifying HIV-1 RNA using the polymerase chain reaction on cerebrospinal fluid and serum of seropositive individuals with and without neurologic abnormalities.

We quantified HIV-1 RNA levels (copies per milliliter) in cerebrospinal fluid (CSF) and serum from subjects at various stages of HIV-1 disease and determined the relationship of RNA levels to clinical and neurologic disease status (HND) and to laboratory values. Ninety-seven HIV-1-seropositive men without CNS opportunistic infections, tumors, or neurosyphilis and 13 high-risk seronegative controls were included in the study. Each individual underwent a structured interview and physical and neurologic examinations, followed by standardized collection of blood and CSF. A custom-designed, fully automated polymerase chain reaction (PCR) system was used to perform a minimum of four separate amplifications per specimen, using two HIV-1 gag primer pairs. Southern blotting followed by hybridization with product-specific probes was used for post-PCR detection. The number of copies per milliliter was determined by relating unknowns to a built-in dilution-series standard curve using an image analysis system. HIV-1 RNA was detectable in 96% of the sera, 78% of the concentrated CSF samples, and 54% of the unconcentrated CSF samples. Serum RNA levels were significantly higher than in CSF. Serum RNA levels were significantly inversely correlated with CD4+ cell counts (p = -0.34; p = 0.03): i.e., higher RNA levels in seropositive subjects were associated with lower numbers of CD4+ cells. Serum RNA levels correlated positively with number of AIDS-related symptoms, dysfunction scores for total neurological examination, mental status score, cranial nerve score, and CNS motor signs score. Serum RNA levels did not correlate significantly with length of time on zidovudine therapy, intrathecal IgG synthesis rate, or albumin leakage. RNA levels in CSF significantly correlated only with intrathecal IgG synthesis rate and with serum RNA levels. These results confirm that serum levels of HIV-1 RNA correlate with HND and inversely correlate with CD4 counts, demonstrating that HND occurs predominantly in late stages of HIV-1 disease, although HIV-1 RNA can be detected in CSF from a majority of HIV-1-seropositive individuals at all stages of disease, which suggests that there can be early penetration of HIV into the CNS. However, HND can occur in the absence of high levels of CSF HIV-1 RNA. We also found that the concentration of HIV-1 in CSF is correlated with intrathecal IgG synthesis rate.