RNA Polymerase Chain Reaction Research Articles

Identification of Blastocystis Subtype 1 Variants in the Home for Girls, Bangkok, Thailand

A cross-sectional study of Blastocystis infection was conducted to evaluate the prevalence, risk factors, and subtypes of Blastocystis at the Home for Girls, Bangkok, Thailand in November 2008. Of 370 stool samples, 118 (31.9%) were infected with Blastocystis. Genotypic characterization of Blastocystis was performed by polymerase chain reaction and sequence analysis of the partial small subunit ribosomal RNA (SSU rRNA) gene. Subtype 1 was the most predominant (94.8%), followed by subtype 6 (3.5%) and subtype 2 (1.7%). Sequence analyses revealed nucleotide polymorphisms for Blastocystis subtype 1, which were described as subtype 1/variant 1, subtype 1/variant 2. Blastocystis subtype 1/variant 1 was the most predominant infection occurring in almost every house. The results showed that subtype analysis of Blastocystis was useful for molecular epidemiological study.

An efficient method for high-fidelity messenger RNA amplification from a small amount of total RNA

Comprehensive analyses of gene expression have been carried out by the development of microarrays and deep sequencers. However, it is difficult to obtain comprehensive information on gene expression from a small amount of ribonucleic acid (RNA). Therefore, we investigated the reproducibility and application of T7 RNA polymerase-mediated transcription, adaptor ligation and polymerase chain reaction (PCR) amplification, followed by T7 transcription (TALPAT), an efficient method for amplifying poly (A)-positive RNA, such as messenger RNA (mRNA). When amplified complementary RNA (cRNA) was electrophoresed, a large number of amplified cRNA was detected in the size of 0.2-0.5 kb. This indicates that the region up to 0.2-0.5 kb from the 3' end of the original mRNA was amplified by the TALPAT method. Seven housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), succinate dehydrogenase complex (SDHA), TATA box-binding protein (TBP) and ubiquitin C (UBC), showed high reproducibility (square of the correlation coefficient, R2=0.9954), according to scatter plots of Ct values obtained in the real-time PCR analysis of amplified cRNA. In addition, relative expression ratios of amplified cRNA of the seven housekeeping genes were approximately equal to the ratio of the original RNA solution. Furthermore, cRNA was amplified from 20 pg total RNA. In the present study, we confirmed the characteristics of mRNA amplification using the TALPAT method. This method may be applicable to mRNA and poly (A)-positive non-coding RNA amplification, using a small amount of RNA from single, laser-captured and sorted cells, as well as exosomes from serum, urine and body fluids.

Performance of HIV-1 DNA or HIV-1 RNA Tests for Early Diagnosis of Perinatal HIV-1 Infection During Anti-retroviral Prophylaxis

M Burgard, S Blanche, C Jasseron. J Pediatr. 2012;160(1):60–66.e1 The identification of HIV-derived nucleic acid in the blood of perinatally exposed infants is the most sensitive and specific method for the early detection of HIV infection in this population. HIV DNA is measured in peripheral blood mononuclear cells and HIV RNA in the plasma. This study compares the performance of HIV DNA polymerase chain reaction (PCR) and HIV RNA PCR for the diagnosis of HIV infection in exposed infants. A total of 1567 children, …

Treatment of Microsporidial Keratoconjunctivitis With Repeated Corneal Swabbing

To report the effect of repeated corneal swabbing in patients with microsporidial keratoconjunctivitis. Retrospective noncomparative case series. Sixteen eyes of 14 healthy patients with microsporidial keratoconjunctivitis were diagnosed based on the detection of microsporidia in corneal scrapings using Gram stain, the modified Kinyoun acid-fast stain, or both. Polymerase chain reaction plus gene analysis of the microsporidian 16S ribosomal RNA had been performed in 10 patients who sought treatment between 2010 and 2011. Some of the lesions were scraped for procurement of specimens. The remaining lesions were wiped off gently by cotton swabs. Repeated swabbing was performed if infection persisted or new lesions were observed at follow-up. To prevent secondary bacterial infection, 0.3% norfloxacin or 0.25 % chloramphenicol were prescribed. The mean age was 52.2 years. All patients had the characteristic disseminated, punctate, slightly elevated, white epithelial lesions. The denser white lesions could be removed easily after gentle swabbing, and most epithelium remained intact. The 10 cases with positive polymerase chain reaction results were all identified to be Vittaforma corneae. The mean number of corneal swabbing was 3.3, and the mean disease resolution time was 6.6 days. No patients had recurrence or loss of visual acuity at last follow-up. Repeated swabbing effectively can eradicate corneal epithelial microsporidial lesions in approximately 1 week. It is easy to perform, less painful, and more acceptable for the patients.

Non‐cirrhotic portal hypertension in HIV mono‐infected patients

Unexplained liver injury including fibrosis and portal hypertension has rarely been reported among patients with HIV in the absence of co-infection with hepatitis B (HBV) or hepatitis C (HCV). We describe a series of HIV mono-infected patients with evidence of non-cirrhotic portal hypertension. HIV-infected patients with evidence of portal hypertension who were anti-HBV and anti-HCV negative and HBV and HCV RNA polymerase chain reaction (PCR) negative were identified from patients managed by the Victorian statewide HIV referral service located at The Alfred Hospital, Melbourne. Portal hypertension was defined as either radiological or endoscopic evidence of varices, portal vein flow obstruction, or elevated hepatic venous pressure gradient (HPVG). Five patients were found to have portal hypertension. These patients were male, aged 41 to 65 years, with known duration of HIV infection between 11 to 25 years. All had been treated with antiretroviral therapy, including didanosine. Tests for metabolic, autoimmune, and hereditary causes of liver disease failed to establish an etiology for the liver injury. All had radiological or endoscopic findings of varices, and four patients had radiological features of portal vein obstruction or flow reversal. Only one patient underwent HPVG measurement, which was elevated. Non-invasive fibrosis assessment revealed increased liver stiffness in three (out of four) patients, and no cirrhotic features were found on those who underwent liver biopsy. To our knowledge, this is the largest published series of non-cirrhotic portal hypertension in HIV mono-infected patients in Australia. Further research is needed to understand what relationship, if any, HIV or its treatments might have on liver injury over time.

Can Broad-Range 16S Ribosomal Ribonucleic Acid Gene Polymerase Chain Reactions Improve the Diagnosis of Bacterial Meningitis? A Systematic Review and Meta-analysis

We aim to evaluate the accuracy of the broad-range 16S polymerase chain reaction test in the diagnosis of bacterial meningitis through a systematic review and meta-analysis. We searched MEDLINE, EMBASE, and the Cochrane Controlled Trials Registry, using the Medical Subject Headings terms "polymerase chain reaction," "RNA, ribosomal, 16S," and "bacterial meningitis." For our primary analysis, we examined the 16S polymerase chain reaction in culture-proven bacterial meningitis. In ancillary observations, we included studies of culture-negative presumed bacterial meningitis, in which there was high clinical suspicion for bacterial meningitis despite negative cerebrospinal fluid culture results. We extracted information necessary to calculate sensitivity and specificity and used bivariate hierarchic modeling meta-analysis methods to obtain pooled statistics. We also estimated potential sources of error and bias such as between-study heterogeneity and publication bias. Fourteen of 299 studies met inclusion criteria for culture-proven bacterial meningitis; 448 (16.1%) of 2,780 subjects had positive cerebrospinal fluid culture results. Pooled analysis demonstrated a sensitivity of 92% (95% confidence interval [CI] 75% to 98%), specificity of 94% (95% CI 90% to 97%), positive likelihood ratio of 16.26 (95% CI 9.07 to 29.14), and negative likelihood ratio of 0.09 (95% CI 0.03 to 0.28) for culture-proven bacterial meningitis. The polymerase chain reaction test result was also positive in 30% of cases of culture-negative presumed bacterial meningitis. There was significant heterogeneity between studies. This meta-analysis supports the role of 16S ribosomal ribonucleic acid polymerase chain reaction as a diagnostic tool in bacterial meningitis. With further refinements in technology, the polymerase chain reaction test has the potential to become a useful adjunct in the diagnosis of bacterial meningitis in the emergency department.

16S RNA PCR in the diagnosis of bacterial keratitis and endophthalmitis

AbstractPurpose Classical bacterial culture often fails to detect the infecting agent in bacterial keratitis and endophthalmitis. Our study was designed to evaluate the contribution of 16S RNA amplification using polymerase chain reaction (PCR) and nested PCR, when compared to culture, in the diagnosis of bacterial keratitis and endophthalmitis.Methods In this prospective study, we included 63 patients (24 bacterial keratitis and 39 endophthalmitis). For every patient, two samples were taken: one for bacterial culture, the other for PCR. PCR and nested PCR 16S rRNA were performed using universal primers (8/27‐1510 for PCR and 91E‐13BS for nested PCR) and the amplicons were thereafter sequenced.Results Culture offers a sensibility of 25%; associated to first PCR (8/27‐1510), this rate increase to 31,7%, and with nested PCR (91E‐13BS), we had 34,9% of identifications. We have to note that the rate of positive PCR were much better, but the sequencing rate was 59,5% only. For bacterial keratitis, culture was positive in 25% and PCR in 8%. For aqueous humor samples, culture was positive in 32%, PCR in 25% and nested PCR in 20,6%. Sequencing was possible in 76,5% for humor aqueous samples.. For vitreous samples, the culture alone was positive in 9%, PCR in 36,3%, and nested PCR in 36,3%.Conclusion 16S rRNA PCR is a rapid alternative to culture, sensitive and reproductible.. PCR associated to culture increases the number of positive samples. PCR was found necessary for the microbiological diagnosis in 10% of all cases (PCR+, cultures‐).

Chlamydiaceae family, Parachlamydia spp., and Waddlia spp. in porcine abortion

At present, despite extensive laboratory investigations, most cases of porcine abortion remain without an etiological diagnosis. Due to a lack of recent data on the abortigenic effect of order Chlamydiales, 286 fetuses and their placentae of 113 abortion cases (1-5 fetuses per abortion case) were investigated by polymerase chain reaction (PCR) methods for family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia acanthamoebae and Waddlia chondrophila. In 0.35% of the cases (1/286 fetuses), the Chlamydiaceae real-time PCR was positive. In the Chlamydiaceae-positive fetus, Chlamydia abortus was detected by a commercial microarray and 16S ribosomal RNA PCR followed by sequencing. The positive fetus had a Porcine circovirus-2 coinfection. By the Parachlamydia real-time PCR, 3.5% (10/286 fetuses of 9 abortion cases) were questionable positive (threshold cycle values: 35.0-45.0). In 2 of these 10 cases, a confirmation by Chlamydiales-specific real-time PCR was possible. All samples tested negative by the Waddlia real-time PCR. It seems unlikely that Chlamydiaceae, Parachlamydia, and Waddlia play an important role as abortigenic agents in Swiss sows.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Disseminated bread yeast fungaemia in a baker's wife with acute myeloid leukaemia

A 58-year-old baker's wife was diagnosed with acute myeloid leukaemia. Induction therapy was initiated with idarubicin, cytarabine and clofarabine (Dutch Trial Registry #NTR2187), with concomitant prophylactic use of ciprofloxacin, trimethoprim-sulfamethoxazole, valaciclovir and amphotericin B oral suspension. Routine surveillance cultures from the oral cavity were positive for fluconazole-sensitive yeasts, but no other commensal microorganisms. Soon after induction treatment, the patient developed abdominal pain and febrile neutropenia. Abdominal ultrasound examination identified multiple hypodense lesions in the liver (left) and the spleen. A liver biopsy was performed, showing fungal micro-organisms (right, periodic acid-Schiff-diastase stain). Polymerase chain reaction for fungal 18S ribosomal RNA was performed and subsequent sequencing identified Saccharomyces cerevisiae, also known as baker's yeast. In our patient, work-related intestinal colonization most likely preceded baker's yeast invasion of the intestinal wall during chemotherapy-induced neutropenic enterocolitis, eventually leading to fungaemia. About 1% of patients with haematological malignancies are colonized with S. cerevisiae at diagnosis. Symptomatic S. cerevisiae infections are rare, but have been described in critically ill and immunocompromised patients. The combination of neutropenic enterocolitis and compromised cellular immunity after clofarabine treatment may have put our patient at an increased risk of developing symptomatic fungaemia. After failure of empirical treatment with voriconazole, she was successfully treated with caspofungin.

HIV-2 Encephalitis: Case Report and Literature Review

We report the case of a 59-year-old man who moved from Cape Verde to Massachusetts at the age of 29. He had multiple sexual contacts with female partners in Cape Verde and with West African women in Massachusetts, as well as multiple past indeterminate HIV-1 antibody tests. He presented to our facility with 2-3 months of inappropriate behaviors, memory impairment, weight loss, and night sweats, at which time he was found to have an abnormal enhancing lesion of the corpus collosum on brain magnetic resonance imaging (MRI). Laboratory testing revealed a CD4 count of 63 cells/mm(3), positive HIV-2 Western blot, serum HIV-2 RNA polymerase chain reaction (PCR) of 1160 copies per milliliter and cerebrospinal fluid (CSF) HIV-2 RNA PCR of 2730 copies per milliliter. Brain biopsy demonstrated syncytial giant cells centered around small blood vessels and accompanied by microglia, which correlated with prior pathologic descriptions of HIV-2 encephalitis and with well-described findings of HIV-1 encephalitis. Based on genotype resistance assay results, treatment guidelines, and prior studies validating success with lopinavir-ritonavir, he was treated with tenofovir-emtricitabine and lopinavir-ritonavir, which has led to virologic suppression along with steady neurologic and radiologic improvement, although he continues to have deficits.

Autophagy modulates osteoarthritis‐related gene expression in human chondrocytes

Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study was to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA). Autophagy in articular cartilage and primary chondrocytes was assessed using antibodies for the autophagy markers light chain 3 and beclin 1. The states of autophagy under catabolic and nutritional stresses were examined. We also examined the effects of inhibition or induction of autophagy under stimulation with interleukin-1β. Autophagy was inhibited by small interfering RNA targeting ATG5, and autophagy was induced by rapamycin. The effects of inhibition or induction of autophagy were examined by real-time polymerase chain reaction for aggrecan, COL2A1, MMP13, and ADAMTS5 messenger RNA. To further examine the mechanism of autophagy regulation in OA human chondrocytes, we investigated whether autophagy modulates apoptosis and reactive oxygen species (ROS). Autophagy was increased in OA chondrocytes and cartilage. Catabolic and nutritional stresses increased autophagy. In addition, the inhibition of autophagy caused OA-like gene expression changes, while the induction of autophagy prevented them. Furthermore, the inhibition of autophagy increased the amount of cleaved poly(ADP-ribose) polymerase and cleaved caspase 9, while the induction of autophagy inhibited these increases. ROS activity was also decreased by induction of autophagy. These observations suggested that increased autophagy is an adaptive response to protect cells from stresses, and that autophagy regulates OA-like gene expression changes through the modulation of apoptosis and ROS. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

Observational study on EFFECT OF HIV INFECTION ON PRESENTATION AND OUTCOME OF diarrhoeal diseases in children less than 5 years of age in Kwazulu-Natal, South Africa

Objectives Primary objective was to compare mortality directly attributed to diarrhoeal diseases in HIV-infected, HIV-exposed but uninfected, and HIV-unexposed children between 2 months and 5 years of age. Secondary objectives were to compare presence of co-morbidities, parameters indicative of severe diarrhoea at presentation and outcome parameters. Methods Intramuscular Triamcinolone for Difficult Asthma Prospective observational study using medical records. 120 children in the appropriate age group were admitted as non-referral cases to Ngwelezane hospital with diarrhoeal diseases between 1st March and 30th June 2011. Children who received traditional remedies prior to admission were excluded (n=28). For 4 children medical records were untraceable. HIV status is confirmed by viral RNA Polymerase Chain Reaction in children younger than 18 months, and either 2 positive rapid tests (different brands) or one positive rapid and one positive ELISA in children older than 18 months. HIV-exposed is confirmed mainly by mother9s Prevention of Mother To Child Transmission status. If this is not available, mother9s Voluntary Counselling Test result at the time of admission is used. Survival analysis and cox regression test were performed on the primary objective. Results Of 88 children, 25 are HIV-positive (28%), 25 HIV-exposed but uninfected (28%) and 38 HIV-unexposed (44%). Mortality directly attributed to diarrhoeal diseases was 6/25 (24%, p=0.08), 4/25 (16%, p=0.19), 2/38 (5%) respectively. Average durations of inpatient-stay are 22, 15 and 12 days respectively. With the exception of presence of dysentery and renal impairment, parameters indicative of severe diarrhoea (severity of dehydration and arterial blood gas results) are similar in all 3 groups. For HIV-positive children, average CD4 count is 462, CD4% 15.5 and viral load 1772720. Conclusions Although not statistically significant at 95% CI, mortality due to diarrhoeal diseases appear higher in HIV-positive children and it occurs more frequently in the first 5 days of admission. This should alert clinicians that HIV-positive children with diarrhoeal diseases require more intensive monitoring and treatment even if their initial presentations are similar to HIV-unexposed children.

Point of care testing of HIV in children younger than 18 months with three different HIV virological assays. Experience from a district hospital in a resource-limited setting

Diagnosis of HIV in children younger than 18 months can be challenging in developing countries because requires the use of HIV virological tests. In this study we describe the experience with three commercial assays, HIV- 1 DNA polymerase chain reaction (PCR) (Roche Amplicor 1.5) with dried blood spot, HIV-1 RNA PCR (Roche COBAS TaqMan) with plasma and reverse transcriptase activity assay (Cavidi Exavir Load 3) with plasma in a rural setting of India. Sensitivity and specificity were 98.1% (95% confidence interval [CI] 90- 100) and 99.3% (95% CI 97.9-99.9) for the HIV- 1 RNA PCR assay, 66.7% (95% CI 29.9-92.5) and 100% (95% CI 96.8-100) for the HIV-1 DNA PCR assay, and 100% (95% CI 48-100) and 98.7% (95% CI 92.8-100) for the reverse transcriptase activity assay respectively. The low sensitivity of the HIV-1 DNA PCR assay in this setting is worrisome and warrants further investigations.

Leucine‐rich repeat kinase 2 and alternative splicing in Parkinson's disease

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD) and are associated with pleiomorphic neuropathology. We hypothesize that LRRK2 mediates its pathogenic effect through alternative splicing of neurodegeneration genes. Methods used in this study included western blotting analysis of subcellular protein fractions, exon-array analysis of RNA from cultured neuroblastoma cells transfected with LRRK2 expression vectors, and reverse-transcription polymerase chain reaction (RT-PCR) of RNA from cultured cells and postmortem tissue. Overexpression of the LRRK2 G2019S mutant resulted in a significant (2.6-fold; P = 0.020) decrease in nuclear transactive response DNA-binding protein 43 levels. Exon-array analyses revealed that wild-type LRRK2 had a significant effect on the expression of genes with nuclear (P < 10(-22) ) and cell-cycle functions (P < 10(-15) ). We replicated changes in gene expression in 30% of selected genes by quantitative RT-PCR. Overexpression of LRRK2 resulted in the altered splicing of two genes associated with PD, with an increased inclusion of exon 10 of microtubule-associated protein tau (1.7-fold; P = 0.001) and exon 5 of the alpha-synuclein (SNCA) gene (1.6-fold; P =0.005). Moreover, overexpression of LRRK2 (G2019S) and two mutant genes associated with neurodegeneration, TARDBP (M337V) and FUS (R521H), were associated with decreased inclusion out of the dystonin (DST) 1e precursor exons in SK-N-MC cells. Altered splicing of SNCA (1.9-fold; P < 0.001) and DST genes (log(2) 2.3-fold; P = 0.005) was observed in a cohort of PD, compared with neurologically healthy, brains. This suggests that aberrant RNA metabolism is an important contributor to idiopathic PD.

Hepatitis E virus-associated aplastic anaemia: The first case of its kind

Here we report the case of a 32-year-old Pakistani male, who developed severe aplastic anaemia after a severe attack of acute hepatitis E virus (HEV) infection. His laboratory test values were not in normal ranges. The liver enzymes were elevated. Serologic and/or molecular-based tests for hepatitis A, B, C, D, G, transfusion transmitted virus (TTV) and B19 were negative, whereas anti-HEV IgM and HEV RNA polymerase chain reaction (PCR) was also detected in the patient sample. The patient received immunosuppressive therapy for 6 months; however, he did not show response to this kind of therapy. The results of our case clearly show the causative role of HEV in the development of aplastic anaemia that might not be ignored.

Survey of the feline leukemia virus infection status of cats in Southern Germany.

Most studies that investigate the prevalence of infections with feline leukemia virus (FeLV) are based on the detection of p27 antigen in blood, but they do not detect proviral DNA to identify the prevalence of regressive FeLV infections. The aim of the present study was to assess the prevalence and status of FeLV infection in cats in Southern Germany. P27 antigen enzyme-linked immunosorbent assay (ELISA), anti-p45 antibody ELISA, DNA polymerase chain reaction (PCR) of blood and RNA PCR of saliva were performed. Nine out of 495 cats were progressively (persistently) infected (1.8%) and six were regressively (latently) infected (1.2%). Cats with regressive infections are defined as cats that have been able to overcome antigenemia but provirus can be detected by PCR. Twenty-two unvaccinated cats likely had abortive infections (regressor cats), testing FeLV antigen- and provirus-negative but anti-p45 antibody-positive. Most of the FeLV-vaccinated cats did not have anti-FeLV antibodies. Both progressive, as well as regressive infections seem to be rare in Germany today.

Forkhead box P1 overexpression and its clinicopathologic significance in peripheral T-cell lymphoma, not otherwise specified

Forkhead box P1 protein is a transcription factor involved in cell signaling and regulation of gene expression and is essential for B-cell development. Forkhead box P1 overexpression has been associated with a worsened prognosis in some B-cell lymphomas. However, little is known about the clinicopathologic significance of forkhead box P1 in T-cell malignancies. In this study, immunohistochemistry for forkhead box P1 was performed in peripheral T-cell lymphoma, not otherwise specified, cases (n = 41), which were then divided into lower (n = 15) and higher (n = 26) forkhead box P1 expressers. Results of real-time quantitative reverse transcriptase polymerase chain reaction for forkhead box P1 messenger RNA supported the data on immunohistochemical forkhead box P1 expression. Forkhead box P1 overexpression in lymphoma cells was inversely associated with proliferation activity as evaluated by Ki-67 expression. Double immunostain for forkhead box P1 and a T-cell marker in normal lymph nodes showed forkhead box P1 signals in many of nonneoplastic T cells. Prognostic analysis showed that forkhead box P1 overexpression was associated with an improved overall survival of the patients with peripheral T-cell lymphoma, not otherwise specified, and was independent of the International Prognostic Index in multivariate analysis. Forkhead box P1 overexpression may be associated with less activated phenotype of the tumors and with a better prognosis in patients with peripheral T-cell lymphoma, not otherwise specified. The clinicopathologic significance of forkhead box P1 overexpression in peripheral T-cell lymphoma, not otherwise specified, may be different from that in B-cell lymphomas.

Real-Time Droplet DNA Amplification with a New Tablet Platform

We present a novel droplet-based tablet platform for temporal polymerase chain reaction (PCR) in microliter droplets. The simple design of the device does not require extensive processing or external equipment, which allows for greater ease of use and integration as a point-of-care diagnostic. We demonstrate its functionality to perform both PCR and reverse-transcription PCR for λ phage DNA and H3 influenza RNA with ramp rates and cycle times consistent with traditional PCR thermal cyclers. We additionally investigate the effect of performing PCR in small volumes on the reaction performance by specifically examining adsorption of reagents at the oil/water interface. We determined that adsorption of Taq polymerase at the biphasic interface reduces yield and impairs reaction performance at standard concentrations. Thus, microdroplet PCR reactions require additional polymerase to achieve sufficient amplification and we project for applications utilizing nanodroplets or picodroplets like digital applications, even greater concentrations of polymerase are required to achieve desired results. Following the adsorption investigation, we evaluated the sensitivity of λ phage PCR on our platform to be less than 2.0 copies/μL with an efficiency of 104.4% and similar sensitivity for reverse-transcription PCR for influenza H3 RNA.

False-positive HIV DNA PCR testing of infants: Implications in a changing epidemic

To examine false-positive HIV DNA polymerase chain reaction (PCR) test results in children, and the potential implications for the paediatric HIV epidemic in sub-Saharan Africa. A review was done of records over a 6-year period of children less than 18 months old at an HIV treatment site in South Africa, to evaluate those with an initial 'false'-positive HIV DNA PCR result, but later proven to be HIV-uninfected with HIV DNA PCR and/or quantitative HIV RNA PCR tests. We calculated the influence of changing HIV transmission rates on predictive values (PV) of HIV DNA PCR tests in a hypothetical population of all HIV-exposed infants over a 1-year period. (Positive PV: proportion of individuals with a positive test with disease; negative PV: proportion of individuals with negative test and no disease). Of 718 children, 40 with an initial positive HIV DNA PCR test were subsequently proven to be HIV-uninfected, resulting in a positive PV of 94.4%. Most (75%) uninfected children had PMTCT interventions and were asymptomatic or mildly symptomatic (77.5%). Calculations using a test specificity of 99.4%, as reported previously, show a decrease in positive PV using a single-test strategy from 98.6% at 30% HIV transmission rate, to 94.8% at 10% transmission, to 62.5% at 1% transmission. Reduction in test specificity further decreases positive PV at low transmission rates. Decreasing mother-to-child HIV transmission rates reduce the positive predictive value of a single HIV DNA PCR test result, necessitating adaptations to diagnostic algorithms to avoid misdiagnosis and inappropriate treatment, especially with early initiation of antiretroviral therapy in asymptomatic infants.