Browsing by Subject "Polymerase chain reaction"
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- ItemOpen Access9β Polymorphism of the Glucocorticoid Receptor Gene Appears to Have Limited Impact in Patients with Addison’s Disease(Public Library of Science, 2014) Ross, Ian Louis; Dandara, Collet; Swart, Marelize; Lacerda, Miguel; Schatz, Desmond; Blom, Dirk JacobusBACKGROUND: Addison’s disease (AD) has been associated with an increased risk of cardiovascular disease. Glucocorticoid receptor polymorphisms that alter glucocorticoid sensitivity may influence metabolic and cardiovascular risk factors in patients with AD. The 9β polymorphism of the glucocorticoid receptor gene is associated with relative glucocorticoid resistance and has been reported to increase the risk of myocardial infarction in the elderly. We explored the impact of this polymorphism in patients with AD. Materials and METHODS: 147 patients with AD and 147 age, gender and ethnicity matched healthy controls were recruited. Blood was taken in a non-fasted state for plasma lipid determination, measurement of cardiovascular risk factors and DNA extraction. RESULTS: Genotype data for the 9β polymorphism was available for 139 patients and 146 controls. AD patients had a more atherogenic lipid profile characterized by an increase in the prevalence of small dense LDL (p = 0.003), increased triglycerides (p = 0.002), reduced HDLC (p<0.001) an elevated highly sensitive C-reactive protein (p = 0.01), compared with controls. The 9β polymorphism (at least one G allele) was found in 28% of patients and controls respectively. After adjusting for age, gender, ethnicity, BMI and hydrocortisone dose per metre square of body surface area in patients, there were no significant metabolic associations with this polymorphism and hydrocortisone doses were not higher in patients with the polymorphism. CONCLUSIONS: This study did not identify any associations between the 9β polymorphism and cardiovascular risk factors or hydrocortisone dose and determination of this polymorphism is therefore unlikely to be of clinical benefit in the management of patients with AD.
- ItemOpen AccessThe co-inheritance of alpha-thalassemia and sickle cell anemia is associated with better hematological indices and lower consultations rate in Cameroonian patients and could improve their survival(Public Library of Science, 2014) Rumaney, Maryam Bibi; Bitoungui, Valentina Josiane Ngo; Vorster, Anna Alvera; Ramesar, Raj; Kengne, Andre Pascal; Ngogang, Jeanne; Wonkam, AmbroiseBACKGROUND: Co-inheritance of α-thalassemia was reported to be associated with a delayed age of disease onset among Cameroonian Sickle Cell Anemia (SCA) patients. The present study aimed to explore the correlation between α-thalassemia, hematological indices, and clinical events in these patients. Methods and FINDINGS: We studied 161 Cameroonian SCA patients and 103 controls (59.1% HbAA) with median ages of 17.5 and 23 years. RFLP-PCR was used to confirm SCA genotype and to describe haplotypes in the HBB-like genes cluster. Multiplex Gap-PCR was performed to investigate the 3.7 kb α-globin gene deletions. SNaPshot PCR, capillary electrophoresis and cycle sequencing were used for the genotyping of 10 SNPs in BCL11A , HMIP1/2 , OR51B5/6 and HBG loci, known to influence HbF levels. Generalised linear regression models adjusted for age, sex and SNPs genotypes was used to investigate effects of α-thalassemia on clinical and hematological indices. The median rate of vaso-occlusive painful crisis and hospitalisations was two and one per year, respectively. Stroke was reported in eight cases (7.4%). Benin haplotype was the most prevalent (66.3%; n = 208 chromosomes). Among patients, 37.3% ( n = 60) had at least one 3.7 kb deletion, compared to 10.9% ( n = 6) among HbAA controls (p<0.001). Among patients, the median RBC count increased with the number of 3.7 kb deletions [2.6, 3.0 and 3.4 million/dl, with no, one and two deletions (p = 0.01)]. The median MCV decreased with the number of 3.7 kb deletion [86, 80, and 68fl, with no, one and two deletions (p<0.0001)], as well as median WBC counts [13.2, 10.5 and 9.8×10 9 /L (p<0.0001. The co-inheritance of α-thalassemia was associated with lower consultations rate (p = 0.038). CONCLUSION: The co-inheritance of α-thalassemia and SCA is associated with improved hematological indices, and lower consultations rate in this group of patients. This could possibly improve their survival and explain the higher proportion of α-thalassemia among patients than controls.
- ItemOpen AccessComparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping(Public Library of Science, 2015) Dube, Felix S; van Mens, Suzan P; Robberts, Lourens; Wolter, Nicole; Nicol, Paul; Mafofo, Joseph; Africa, Samantha; Zar, Heather J; Nicol, Mark PBACKGROUND: Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. METHODS: A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. RESULTS: Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A. CONCLUSION: The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.
- ItemOpen AccessComparison of quantitative techniques including Xpert MTB/RIF to evaluate mycobacterial burden(Public Library of Science, 2011) van Zyl-Smit, Richard N; Binder, Anke; Meldau, Richard; Mishra, Hridesh; Semple, Patricia L; Theron, Grant; Peter, Jonathan; Whitelaw, Andrew; Sharma, Suren K; Warren, Robin; Bateman, Eric D; Dheda, KeertanIntroduction: Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens. METHODS: We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [ 3 H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard. RESULTS: All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×10 3 organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p = 0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p = 0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ∼100 organisms. CONCLUSIONS: Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold.
- ItemOpen AccessDetection of Streptococcus pneumoniae from different types of nasopharyngeal swabs in children(Public Library of Science, 2013) Dube, Felix S; Kaba, Mamadou; Whittaker, Elizabeth; Zar, Heather J; Nicol, Mark PBACKGROUND: A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs. METHODS: The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA -targeted real-time polymerase chain reaction (qPCR). RESULTS: Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5-16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10 4 CFU/mL [IQR, 2.0×10 2 - 4.0×10 5 CFU/mL]) than Dacron swabs (3.7×10 4 CFU/mL [IQR, 4.0×10 2 -3.2×10 5 CFU/mL], p = 0.17). Using lytA -targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10 5 genome copies/mL [IQR, 1.3×10 2 −1.8×10 6 ] vs. 9.3×10 4 genome copies/mL [IQR, 7.0×10 1 −1.1×10 6 ]; p = 0.005). CONCLUSION: Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.
- ItemOpen AccessDiagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for tuberculous meningitis in a high burden setting: a prospective study(Public Library of Science, 2013) Patel, Vinod B; Theron, Grant; Lenders, Laura; Matinyena, Brian; Connolly, Cathy; Singh, Ravesh; Coovadia, Yacoob; Ndung'u, Thumbi; Dheda, KeertanBackground: Tuberculous meningitis (TBM) is difficult to diagnose promptly. The utility of the Xpert MTB/RIF test for the diagnosis of TBM remains unclear, and the effect of host- and sample-related factors on test performance is unknown. This study sought to evaluate the sensitivity and specificity of Xpert MTB/RIF for the diagnosis of TBM. Methods and Findings: 235 South-African patients with a meningeal-like illness were categorised as having definite (culture or Amplicor PCR positive), probable (anti-TBM treatment initiated but microbiological confirmation lacking), or non-TBM. Xpert MTB/RIF accuracy was evaluated using 1 ml of uncentrifuged and, when available, 3 ml of centrifuged cerebrospinal fluid (CSF). To evaluate the incremental value of MTB/RIF over a clinically based diagnosis, test accuracy was compared to a clinical score (CS) derived using basic clinical and laboratory information. Of 204 evaluable patients (of whom 87% were HIV-infected), 59 had definite TBM, 64 probable TBM, and 81 non-TBM. Overall sensitivity and specificity (95% CI) were 62% (48%–75%) and 95% (87%–99%), respectively. The sensitivity of Xpert MTB/RIF was significantly better than that of smear microscopy (62% versus 12%; p = 0.001) and significantly better than that of the CS (62% versus 30%; p = 0.001; C statistic 85% [79%–92%]). Xpert MTB/RIF sensitivity was higher when centrifuged versus uncentrifuged samples were used (82% [62%–94%] versus 47% [31%–61%]; p = 0.004). The combination of CS and Xpert MTB/RIF (Xpert MTB/RIF performed if CS<8) performed as well as Xpert MTB/RIF alone but with a ∼10% reduction in test usage. This overall pattern of results remained unchanged when the definite and probable TBM groups were combined. Xpert MTB/RIF was not useful in identifying TBM among HIV-uninfected individuals, although the sample was small. There was no evidence of PCR inhibition, and the limit of detection was ∼80 colony forming units per millilitre. Study limitations included a predominantly HIV-infected cohort and the limited number of culture-positive CSF samples. Conclusions: Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals from a tuberculosis-endemic setting, particularly when a centrifuged CSF pellet is used. Further studies are required to confirm these findings in different settings.
- ItemOpen AccessThe formulation and refinement of a polymerase chain reaction (PCR) assay for early diagnosis of paediatric HIV infection and genetic analysis of variants involved in vertical transmission of HIV-1(1996) Nolte, Jeanine Lucasta; Smuts, Heidi; Stannard, LindaPaediatric human immunodeficiency virus (HIV) infection has become a major socio-economic health problem in recent years as the number of HIV-1 infected children steadily increases. The majority of these infants are infected through mother-to-child transmission, with the frequency of vertical transmission varying between 12,9% and 65%. In order to implement appropriate management and possible treatment of these infected neonates, it is essential to have reliable laboratory tests for the early diagnosis of an HIV infection. At the time that this study was initiated, the diagnosis of HIV-1 infection in the Groote Schuur Hospital Virology Laboratory depended almost exclusively on serological assays. Such assays are of limited value for infants under 18 months of age, as maternal lgG antibody to HIV-1 is transferred via the placenta and may persist in the baby for up to 18 months. Available lgG antibody tests do not distinguish reliably between passively acquired maternal antibody and that produced by the infant itself. A valuable method of establishing the presence of true infection is provided by the polymerase chain reaction (PCR) technique which allows the identification, and subsequent exponential amplification of low levels of specific viral nucleic acid using specific oligonucleotide primers. A major aim of this study was to develop and instigate a (PCR) assay for the early diagnosis of HIV infection in infected infants. This was successfully achieved by the adaptation and optimization of an existing standard PCR protocol to suit the specific needs of a routine diagnostic service. Preliminary requirements involved the selection of primers and probes and establishing optimal parameters for: ionic strength, Taq DNA polymerase concentration, primer concentration, deoxynucleotide triphosphate concentration, and hybridization conditions for most efficient functioning of the test. The devised method entailed the extraction of proviral DNA from peripheral blood mononuclear cells, amplification of HIV-1 specific sequences by PCR, and identification by Southern blot hybridization with digoxigenin (DIG)-labelled probes. Thereafter the efficacy of the assay was tested on 45 infants (under 15 months of age) all born to seropositive mothers and therefore at risk for HIV infection. Forty-two of these infants had antibodies to HIV-1 and the remaining 3 were seronegative. The latter 3 also tested negative for HIV proviral DNA when PCR was performed, using at least 2 different HIV-1 primer pairs and their respective DIG-labelled probes. However, 27 (64%) of the 42 seropositive infants were also HIV-PCR positive and the remaining 15 (36%) seropositive infants were negative for HIV proviral DNA. Positive PCR tests correlated well with clinical data indicative of active HIV-1 infection for the majority of infants in the neonatal period, although it could not provide proof of infection in newborn babies (less than 1 week of age). The development of an in-house PCR protocol specific for HIV-1 has not only provided a valuable diagnostic assay for neonatal infection, but has also given insight into the parameters required for high sensitivity and the stringent precautionary measures that need to be applied to avoid contamination problems. The second part of this study was devoted to DNA sequence analysis of cloned HIV isolates from an infected mother and her 3-month-old infant. Nucleotide sequence variation between isolates of HIV-1 has been well documented. Examination of the third variable region (particularly the V3- loop) in the env gene of HIV-1 of our mother-infant pair confirmed this variation and provided the first genetic epidemiological data of this nature in the local community. Proviral DNA from both mother and baby was amplified using V3-specific degenerate primers and cloned. Clones containing the insert DNA were 2 identified by colony-blot hybridization. Their nucleotide and amino acid sequences were analyzed by using various computer programs. The degree of similarity between variants from the mother and infant in this study differed to a large extent from previous studies. The virus population harboured by the mother displayed highly homogeneous V3 sequences (1,04% variation) compared to the isolates from her 3-month-old infant, which showed a higher degree (1,8%) of heterogeneity. Phylogenetic analysis of the different isolates from mother and infant demonstrated that an HIV-1 subtype C virus was the infectious agent. This classification was confirmed by the characteristic amino-acid sequence of the tetrapeptide motif of the V3 loop present in the isolates from both mother and infant as well as the absence of a potential N-linked glycosylation site proximal to the first cysteine of the V3 loop, which is characteristic of subtype C viruses. Based on the amino acids present at positions 306 and 320 of the V3 loop, it could also be concluded that isolates from both the mother and her baby were consistent with the non-syncytium inducing (NSI) phenotype of HIV-1, thus indicating that, contrary to popular belief, NSI variants can be responsible for initiating infection. Data obtained from these genetic investigations of variants involved in vertical transmission of HIV-1 can form a useful basis for future comparative studies.
- ItemOpen AccessgbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples(Public Library of Science, 2015) Vezzulli, Luigi; Stauder, Monica; Grande, Chiara; Pezzati, Elisabetta; Verheye, Hans M.; Owens, Nicholas J. P.; Pruzzo, CarlaThe Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V . cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V . cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V . cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V . cholerae and failed to amplify strains of the closely-related species Vibrio mimicus . The sensitivity of the assay applied to environmental and stool samples spiked with V . cholerae ATCC 39315 was comparable to that of pure cultures and was of 10 2 genomic units/l for drinking and seawater samples, 10 1 genomic units/g for sediment and 10 2 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V . cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V . cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.
- ItemOpen AccessHepatitis B infection, viral load and resistance in HIV-infected patients in Mozambique and Zambia(Public Library of Science, 2016) Wandeler, Gilles; Musukuma, Kalo; Zürcher, Samuel; Vinikoor, Michael J; Llenas-García, Jara; Aly, Mussa M; Mulenga, Lloyd; Chi, Benjamin H; Ehmer, Jochen; Hobbins, Michael A; Bolton-Moore, Carolyn; Hoffmann, Christopher J; Egger, Matthias; IeDEA-Southern AfricaBACKGROUND: Few data on the virological determinants of hepatitis B virus (HBV) infection are available from southern Africa. METHODS: We enrolled consecutive HIV-infected adult patients initiating antiretroviral therapy (ART) at two urban clinics in Zambia and four rural clinics in Northern Mozambique between May 2013 and August 2014. HBsAg screening was performed using the Determine ® rapid test. Quantitative real-time PCR and HBV sequencing were performed in HBsAg-positive patients. Risk factors for HBV infection were evaluated using Chi-square and Mann-Whitney tests and associations between baseline characteristics and high level HBV replication explored in multivariable logistic regression. RESULTS: Seventy-eight of 1,032 participants in Mozambique (7.6%, 95% confidence interval [CI]: 6.1-9.3) and 90 of 797 in Zambia (11.3%, 95% CI: 9.3-13.4) were HBsAg-positive. HBsAg-positive individuals were less likely to be female compared to HBsAg-negative ones (52.3% vs. 66.1%, p<0.001). Among 156 (92.9%) HBsAg-positive patients with an available measurement, median HBV viral load was 13,645 IU/mL (interquartile range: 192-8,617,488 IU/mL) and 77 (49.4%) had high values (>20,000 UI/mL). HBsAg-positive individuals had higher levels of ALT and AST compared to HBsAg-negative ones (both p<0.001). In multivariable analyses, male sex (adjusted odds ratio: 2.59, 95% CI: 1.22-5.53) and CD4 cell count below 200/μl (2.58, 1.20-5.54) were associated with high HBV DNA. HBV genotypes A1 (58.8%) and E (38.2%) were most prevalent. Four patients had probable resistance to lamivudine and/or entecavir. CONCLUSION: One half of HBsAg-positive patients demonstrated high HBV viremia, supporting the early initiation of tenofovir-containing ART in HIV/HBV-coinfected adults.
- ItemOpen AccessHepatitis C virus infection rate in volunteer blood donors from the Western Cape : comparison of screening tests and PCR(1997) Tucker, TJ; Voigt, M; Bird, A; ROBSON, S; Gibbs, B; KANNEMEYER, J; Galloway, M; Kirsch, AE; SMUTS, HINTRODUCTION: Hepatitis C virus (HCV) antibody seroprevalence studies overestimate the true infection rate. No data exist on the incidence of HCV or its clinical features in blood donors of sub-Saharan Africa. AIMS: To establish the true incidence of HCV infection in volunteer blood donors in the Western Cape, and compare risk factors and clinical and biochemical features of viraemic and non-viraemic subjects. METHODS: All donors attending the Western Province Blood Transfusion Service between December 1992 and August 1994 were screened prospectively for anti-HCV using the Abbott second-generation assay. Positive donors were evaluated clinically and biochemically. Their sera were examined for HCV-RNA by the polymerase chain reaction (PCR). RESULTS: Of 66314 donors screened, 275 (0.41%) were anti-HCV-positive. Of these 13.6% were PCR-positive (0.056% of all donors). PCR-positive patients had more risk factors for HCV acquisition (P < 0.01), symptoms of hepatitis (P = 0.02) and clinical signs of liver disease (P = 0.05) and higher alanine (P < 0.0001) and aspartate aminotransferase levels (P < 0.0001) than PCR-negative donors. However, clinical and biochemical features did not discriminate adequately between PCR-positive and negative donors. Liver biopsies performed in 9 of 13 PCR-positive cases showed mild inflammation, but no cirrhosis.
- ItemOpen AccessHeterozygosity for a hypomorphic polβ mutation reduces the expansion frequency in a mouse model of the fragile x-related disorders(Public Library of Science, 2015) Lokanga, Rachel Adihe; Senejani, Alireza Ghodsi; Sweasy, Joann Balazs; Usdin, KarenAuthor Summary Unstable microsatellites are responsible for a number of debilitating human diseases known as the Repeat Expansion Diseases. The unstable microsatellites, which consist of tandem arrays of short repeat units, are prone to increase in length (expand) on intergenerational transmission and during the lifetime of the individual. Unlike the typical microsatellite instability seen in disorders like Lynch syndrome that arise from mutations in mismatch repair (MMR) genes, expansions of these microsatellites are abolished when MMR is lost. However, how MMR, which normally protects the genome against microsatellite instability, actually promotes microsatellite expansions in these diseases is unknown. There is evidence to suggest that a second DNA repair process, base excision repair (BER), may be involved, but whether the nicks generated early in the BER-process are subverted by an MMR-dependent pathway that generates expansions or whether some MMR proteins contribute to a BER-based expansion process is unclear. Here we show that a mutation that reduces the activity of Polβ, an essential BER enzyme, also reduces the expansion frequency. Since Polβ is essential for key events in BER downstream of the generation of nicks, our data favor a model in which expansions occur via a BER-dependent pathway in which MMR participates.
- ItemOpen AccessHIV-1 subtype C mosaic Gag expressed by BCG and MVA elicits persistent effector t cell responses in a prime-boost regimen in mice(Public Library of Science, 2016) Jongwe, Tsungai Ivai; Chapman, Ros; Douglass, Nicola; Chetty, Shivan; Chege, Gerald; Williamson, Anna-LiseOver 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C) viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG Δ panCD auxotroph and modified vaccinia Ankara (MVA) vaccines expressing HIV-1C mosaic Gag (Gag M ) were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-Gag M vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-Gag M and boosting with MVA-Gag M elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-Gag M only and MVA-Gag M only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4 + and CD8 + T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (10 4 pfu) can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C).
- ItemOpen AccessHost specificity and co-speciation in avian haemosporidia in the Western Cape, South Africa(Public Library of Science, 2014) Okanga, Sharon; Cumming, Graeme S; Hockey, Philip A R; Nupen, Lisa; Peters, Jeffrey LHost and pathogen ecology are often closely linked, with evolutionary processes often leading to the development of host specificity traits in some pathogens. Host specificity may range from ‘generalist’, where pathogens infect any available competent host; to ‘specialist’, where pathogens repeatedly infect specific host species or families. Avian malaria ecology in the region remains largely unexplored, despite the presence of vulnerable endemic avian species. We analysed the expression of host specificity in avian haemosporidia, by applying a previously developed host specificity index to lineages isolated from wetland passerines in the Western Cape, South Africa. Parasite lineages were isolated using PCR and identified when possible using matching lineages deposited in GenBank and in MalAvi. Parasitic clades were constructed from phylogenetic trees consisting of Plasmodium and Haemoproteus lineages. Isolated lineages matched some strains of Plasmodium relictum , P. elongatum , Haemoproteus sylvae and H. lanii . Plasmodium lineages infected a wide range of hosts from several avian families in a generalist pattern of infection. Plasmodium spp. also exhibited an infection trend according to host abundance rather than host species. By contrast, Haemoproteus lineages were typically restricted to one or two host species or families, and displayed higher host fidelity than Plasmodium spp. The findings confirm that a range of host specificity traits are exhibited by avian haemosporidia in the region. The traits show the potential to not only impact infection prevalence within specific host species, but also to affect patterns of infection at the community level.
- ItemOpen AccessIdentification of a collagen type I adhesin of Bacteroides fragilis(Public Library of Science, 2014) Galvão, Bruna P G V; Weber, Brandon W; Rafudeen, Mohamed S; Ferreira, Eliane O; Patrick, Sheila; Abratt, Valerie RBacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.
- ItemOpen AccessIdentification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis(Public Library of Science, 2009) Ubuka, Takayoshi; Morgan, Kevin; Pawson, Adam J; Osugi, Tomohiro; Chowdhury, Vishwajit S; Minakata, Hiroyuki; Tsutsui, Kazuyoshi; Millar, Robert P; Bentley, George EThe existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH 2 ) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH 2 (human RFRP-1) and VPNLPQRF-NH 2 (human RFRP-3) by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca 2+ mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.
- ItemOpen AccessIncreased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature(Public Library of Science, 2014) Carstens, Maryke; McCrindle, Tyronne K; Adams, Nicolette; Diener, Anastashia; Guzha, Delroy T; Murray, Shane L; Parker, Jane E; Denby, Katherine J; Ingle, Robert AThe Arabidopsis constitutive induced resistance 1 ( cir1 ) mutant displays salicylic acid (SA)-dependent constitutive expression of defence genes and enhanced resistance to biotrophic pathogens. To further characterise the role of CIR1 in plant immunity we conducted epistasis analyses with two key components of the SA-signalling branch of the defence network, ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We demonstrate that the constitutive defence phenotypes of cir1 require both EDS1 and PAD4, indicating that CIR1 lies upstream of the EDS1-PAD4 regulatory node in the immune signalling network. In light of this finding we examined EDS1 expression in cir1 and observed increased protein, but not mRNA levels in this mutant, suggesting that CIR1 might act as a negative regulator of EDS1 via a post-transcriptional mechanism. Finally, as environmental temperature is known to influence the outcome of plant-pathogen interactions, we analysed cir1 plants grown at 18, 22 or 25°C. We found that susceptibility to Pseudomonas syringae pv. tomato ( Pst ) DC3000 is modulated by temperature in cir1 . Greatest resistance to this pathogen (relative to PR-1:LUC control plants) was observed at 18°C, while at 25°C no difference in susceptibility between cir1 and control plants was apparent. The increase in resistance to Pst DC3000 at 18°C correlated with a stunted growth phenotype, suggesting that activation of defence responses may be enhanced at lower temperatures in the cir1 mutant.
- ItemOpen AccessLaboratory evaluation of the Alere q point-of-care system for early infant HIV diagnosis(Public Library of Science, 2016) Hsiao, Nei-yuan; Dunning, Lorna; Kroon, Max; Myer, LandonIntroduction Early infant diagnosis (EID) and prompt linkage to care are critical to minimise the high morbidity and mortality associated with infant HIV infection. Attrition in the "EID cascade" is common; however, point-of-care (POC) EID assays with same-day result could facilitate prompt linkage of HIV-infected infant to treatment. Despite a number of POC EID assays in development, few have been independently evaluated and data on new technologies are urgently needed to inform policy. METHODS: We compared Alere q 1/2 Detect POC system laboratory test characteristics with the local standard of care (SOC), Roche CAP/CTM HIV-1 qualitative PCR in an independent laboratory-based evaluation in Cape Town, South Africa. Routinely EID samples collected between November 2013 and September 2014 were each tested by both SOC and POC systems. Repeat testing was done to troubleshoot any discrepancy between POC and SOC results. RESULTS: Overall, 1098 children with a median age of 47 days (IQR, 42-117) were included. Birth PCR (age <7 days) comprised of 8% (n = 92) tests while 56% (n = 620) of children tested as part of routine EID (ages 6-14 weeks). In the overall direct comparison, Alere q Detect achieved sensitivity of 95.5% (95% CI, 91.7-97.9%) and a specificity of 99.8% (95% CI, 99.1-100%). Following repeat testing of discordant samples and exclusion of any inconclusive results, the POC assay sensitivity and specificity were 96.9% (95% CI 93.4-98.9%) and 100% (lower 95% CI 98%) respectively. Among birth PCR tests the POC assay had slightly lower sensitivity (93.3% vs 96.5% in routine EID) and higher assay error rate (10% vs 5% in samples of older children, p = 0.04). CONCLUSION: Our results indicate this POC assay performs well for EID in the laboratory. The high specificity and thus high positive predictive value would suggest a positive POC result may be adequate for immediate infant ART initiation. While POC testing for EID may have particular utility for birth testing at delivery facilities, the lower sensitivity and error rate requires further attention, as does field implementation of POC EID technologies in other clinical care settings.
- ItemOpen AccessA MutSβ-Dependent Contribution of MutSα to Repeat Expansions in Fragile X Premutation Mice?(Public Library of Science, 2016) Zhao, Xiao-Nan; Lokanga, Rachel; Allette, Kimaada; Gazy, Inbal; Wu, Di; Usdin, KarenAuthor Summary: The repeat expansion diseases are a group of human genetic disorders that are caused by expansion of a specific microsatellite in a single affected gene. How this expansion occurs is unknown, but previous work in various models for different diseases in the group, including the fragile X-related disorders (FXDs), has implicated the mismatch repair complex MutSβ in the process. With the exception of somatic expansion in Friedreich ataxia, MutSα has not been reported to contribute to generation of expansions in other disease models. Here we show that MutSα does in fact play a role in both germ line and somatic expansions in a mouse model of the FXDs since the expansion frequency is significantly reduced in Msh6 -/- mice. However, since we have previously shown that loss of MutSβ eliminates almost all expansions, MutSα is apparently not able to fully substitute for MutSβ in the expansion process. We also show here that MutSα increases the stability of the structures formed by the fragile X repeats that are thought to be the substrates for expansion and promotes binding of MutSβ to the repeats. This, together with our genetic data, suggests possible models for how MutSα and MutSβ, could co-operate to generate repeat expansions in the FXDs.
- ItemOpen AccessNew insights into samango monkey speciation in South Africa(Public Library of Science, 2015) Dalton, Desiré L; Linden, Birthe; Wimberger, Kirsten; Nupen, Lisa Jane; Tordiffe, Adrian S W; Taylor, Peter John; Madisha, M Thabang; Kotze, AntoinetteThe samango monkey is South Africa's only exclusively forest dwelling primate and represents the southernmost extent of the range of arboreal guenons in Africa. The main threats to South Africa's forests and thus to the samango are linked to increasing land-use pressure and increasing demands for forest resources, resulting in deforestation, degradation and further fragmentation of irreplaceable habitats. The species belongs to the highly polytypic Cercopithecus nictitans group which is sometimes divided into two species C . mitis and C . albogularis . The number of subspecies of C . albogularis is also under debate and is based only on differences in pelage colouration and thus far no genetic research has been undertaken on South African samango monkey populations. In this study we aim to further clarify the number of samango monkey subspecies, as well as their respective distributions in South Africa by combining molecular, morphometric and pelage data. Overall, our study provides the most comprehensive view to date into the taxonomic description of samango monkeys in South Africa. Our data supports the identification of three distinct genetic entities namely; C . a . labiatus , C . a . erythrarchus and C . a . schwarzi and argues for separate conservation management of the distinct genetic entities defined by this study.
- ItemOpen AccessOverexpression of Kpnβ1 and Kpnα2 Importin Proteins in Cancer Derives from Deregulated E2F Activity(Public Library of Science, 2011) van der Watt, Pauline J; Ngarande, Ellen; Leaner, Virna DThe Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnβ1 and Kpnα2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnβ1 and Kpnα2 levels in cancer cells. Kpnβ1 (−2013 to +100) and Kpnα2 (−1900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the −637 to −271 Kpnβ1 and −180 to −24 Kpnα2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnβ1 and Kpnα2 promoters in vivo . Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnβ1 and Kpnα2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnβ1 and Kpnα2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnβ1 and Kpnα2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities.