Yes / Polymerase chain reaction (PCR) has become a powerful tool for detecting various diseases due to its high sensitivity and specificity. However, the long thermocycling time and the bulky system have limited the application of PCR devices in Point-of-care testing. Herein, we have proposed an efficient, low-cost, and hand-hold PCR microdevice, mainly including a control module based on water-cooling technology and an amplification module fabricated by 3D printing. The whole device is tiny and can be easily hand-held with a size of about 110 mm × 100 mm × 40 mm and a weight of about 300 g at a low cost of about $170.83. Based on the water-cooling technology, the device can efficiently perform 30 thermal cycles within 46 min at a heating/cooling rate of 4.0/8.1 ℃/s. To test our instrument, plasmid DNA dilutions were amplified with this device; the results demonstrate successful nucleic acid amplification of the …

This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Nucleic acid sequence based amplification (NASBA) is a primer based isothermal method of RNA/DNA amplification. Currently, primer design for NASBA has been restricted to hand creating sequences of oligonucleotides that must follow a set of rules to be compatible for the amplification process. This process of hand-creating primers is prone to error and time intensive. The detection of mutants, post amplification, also offers a benefit in point of care scenarios and the design of hybridization probes for sequences in the region of amplification is also an erroneous and time intensive process. By creating a program to design primers and hybridization probes based on the set of rules provided for a sequence of user input DNA or RNA, one can avoid costly errors in primers design and save time. Utilizing Python (a high-level object-oriented programming language), along with a series of bioinformatic libraries such as Biopython and UNAfold one can definitively choose the best primer sequences for a given sample of DNA.

Introdução: A infecção urogenital por Chlamydia trachomatis é a doença sexualmente transmissível bacteriana mais prevalente no mundo e afeta principalmente mulheres jovens sexualmente ativas. Infecções não tratadas podem provocar complicações reprodutivas decorrentes do dano tubáreo. Na gestação, aumenta o risco de parto prematuro, baixo peso ao nascer, morte perinatal, conjuntivite e pneumonia neonatal. Existem poucos dados brasileiros referentes à epidemiologia dessa infecção no nosso meio. O objetivo desse estudo foi determinar a prevalência de C. trachomatis em mulheres inférteis e em gestantes. Método: Foram analisadas transversalmente 77 mulheres inférteis e 60 gestantes assintomáticas. Foram coletadas amostras urinárias para ensaio de PCR e amostras sanguíneas para pesquisa de anticorpos IgG através da técnica de imunofluorescência indireta. Todas as participantes responderam um questionário referente ao seu histórico clínico e ginecológico. Resultados: A prevalência, tanto no ensaio de PCR quanto na imunofluorescência indireta (IgG) para C. trachomatis foi similar entre os grupos. Encontramos anticorpos IgG presentes em 61% das mulheres inférteis e em 56,7% das gestantes. Houve somente 1 PCR positivo no grupo das inférteis (1,3%) e nenhum do grupo das gestantes. Conclusão: Encontramos alta prevalência de anticorpos IgG para C. trachomatis em mulheres inférteis e em gestantes, mas verificamos baixa prevalência de PCR positivo nas participantes. A presença de IgG correlacionou-se com comportamento sexual e tabagismo. / Background: Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection and affects mainly young, sexually active, women. Untreated infection may lead to reproductive complications due to tubal damage. Infections during pregnancy may cause preterm labor, low birth weight, perinatal death and neonatal conjunctivitis and pneumonia. There is little data on CT infection in Brazil. The aim of this study was to determine CT prevalence on infertile and pregnant women. Methods: A cross-sectional study included 77 infertile and 60 asymptomatic pregnant women. First void urine was tested to CT using PCR and blood samples were collected for CT IgG antibodies testing using Indirect Immunofluorescence. A questionnaire about medical, gynecological and sexual history was applied to all participants. Results: We found statistically similar prevalence of PCR and IgG antibodies between groups. This study observed a 61% prevalence of CT IgG antibodies in infertile women and 56,7% in pregnant women. PCR was positive in only one (1,3%) infertile woman and in none of the pregnant. Conclusion: A high prevalence of C. trachomatis IgG antibody in Brazilian pregnant and infertile women, but a low prevalence of positive PCR on urine samples were demonstrated. CT antibodies were associated with sexual behavior and smoking.

Introdução: A infecção urogenital por Chlamydia trachomatis é a doença sexualmente transmissível bacteriana mais prevalente no mundo e afeta principalmente mulheres jovens sexualmente ativas. Infecções não tratadas podem provocar complicações reprodutivas decorrentes do dano tubáreo. Na gestação, aumenta o risco de parto prematuro, baixo peso ao nascer, morte perinatal, conjuntivite e pneumonia neonatal. Existem poucos dados brasileiros referentes à epidemiologia dessa infecção no nosso meio. O objetivo desse estudo foi determinar a prevalência de C. trachomatis em mulheres inférteis e em gestantes. Método: Foram analisadas transversalmente 77 mulheres inférteis e 60 gestantes assintomáticas. Foram coletadas amostras urinárias para ensaio de PCR e amostras sanguíneas para pesquisa de anticorpos IgG através da técnica de imunofluorescência indireta. Todas as participantes responderam um questionário referente ao seu histórico clínico e ginecológico. Resultados: A prevalência, tanto no ensaio de PCR quanto na imunofluorescência indireta (IgG) para C. trachomatis foi similar entre os grupos. Encontramos anticorpos IgG presentes em 61% das mulheres inférteis e em 56,7% das gestantes. Houve somente 1 PCR positivo no grupo das inférteis (1,3%) e nenhum do grupo das gestantes. Conclusão: Encontramos alta prevalência de anticorpos IgG para C. trachomatis em mulheres inférteis e em gestantes, mas verificamos baixa prevalência de PCR positivo nas participantes. A presença de IgG correlacionou-se com comportamento sexual e tabagismo. / Background: Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection and affects mainly young, sexually active, women. Untreated infection may lead to reproductive complications due to tubal damage. Infections during pregnancy may cause preterm labor, low birth weight, perinatal death and neonatal conjunctivitis and pneumonia. There is little data on CT infection in Brazil. The aim of this study was to determine CT prevalence on infertile and pregnant women. Methods: A cross-sectional study included 77 infertile and 60 asymptomatic pregnant women. First void urine was tested to CT using PCR and blood samples were collected for CT IgG antibodies testing using Indirect Immunofluorescence. A questionnaire about medical, gynecological and sexual history was applied to all participants. Results: We found statistically similar prevalence of PCR and IgG antibodies between groups. This study observed a 61% prevalence of CT IgG antibodies in infertile women and 56,7% in pregnant women. PCR was positive in only one (1,3%) infertile woman and in none of the pregnant. Conclusion: A high prevalence of C. trachomatis IgG antibody in Brazilian pregnant and infertile women, but a low prevalence of positive PCR on urine samples were demonstrated. CT antibodies were associated with sexual behavior and smoking.

Introdução: A infecção urogenital por Chlamydia trachomatis é a doença sexualmente transmissível bacteriana mais prevalente no mundo e afeta principalmente mulheres jovens sexualmente ativas. Infecções não tratadas podem provocar complicações reprodutivas decorrentes do dano tubáreo. Na gestação, aumenta o risco de parto prematuro, baixo peso ao nascer, morte perinatal, conjuntivite e pneumonia neonatal. Existem poucos dados brasileiros referentes à epidemiologia dessa infecção no nosso meio. O objetivo desse estudo foi determinar a prevalência de C. trachomatis em mulheres inférteis e em gestantes. Método: Foram analisadas transversalmente 77 mulheres inférteis e 60 gestantes assintomáticas. Foram coletadas amostras urinárias para ensaio de PCR e amostras sanguíneas para pesquisa de anticorpos IgG através da técnica de imunofluorescência indireta. Todas as participantes responderam um questionário referente ao seu histórico clínico e ginecológico. Resultados: A prevalência, tanto no ensaio de PCR quanto na imunofluorescência indireta (IgG) para C. trachomatis foi similar entre os grupos. Encontramos anticorpos IgG presentes em 61% das mulheres inférteis e em 56,7% das gestantes. Houve somente 1 PCR positivo no grupo das inférteis (1,3%) e nenhum do grupo das gestantes. Conclusão: Encontramos alta prevalência de anticorpos IgG para C. trachomatis em mulheres inférteis e em gestantes, mas verificamos baixa prevalência de PCR positivo nas participantes. A presença de IgG correlacionou-se com comportamento sexual e tabagismo. / Background: Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection and affects mainly young, sexually active, women. Untreated infection may lead to reproductive complications due to tubal damage. Infections during pregnancy may cause preterm labor, low birth weight, perinatal death and neonatal conjunctivitis and pneumonia. There is little data on CT infection in Brazil. The aim of this study was to determine CT prevalence on infertile and pregnant women. Methods: A cross-sectional study included 77 infertile and 60 asymptomatic pregnant women. First void urine was tested to CT using PCR and blood samples were collected for CT IgG antibodies testing using Indirect Immunofluorescence. A questionnaire about medical, gynecological and sexual history was applied to all participants. Results: We found statistically similar prevalence of PCR and IgG antibodies between groups. This study observed a 61% prevalence of CT IgG antibodies in infertile women and 56,7% in pregnant women. PCR was positive in only one (1,3%) infertile woman and in none of the pregnant. Conclusion: A high prevalence of C. trachomatis IgG antibody in Brazilian pregnant and infertile women, but a low prevalence of positive PCR on urine samples were demonstrated. CT antibodies were associated with sexual behavior and smoking.

<p dir="ltr">Since the beginning of the HIV/AIDS epidemic, 85.6 million people worldwide have become infected with HIV; more than half of whom have died from AIDS-related complications.[1] Sustained viral suppression below the clinically relevant threshold (1000 copies per mL) with highly active antiretroviral therapy (HAART) has proven effective at managing and prolonging the life expectancy of people living with HIV (PLHIV). However, in 2022, 11.3 million PLHIV had still not achieved viral suppression and may become susceptible to both HIV transmission and a variety of opportunistic infections. Of particular importance is the complex issue of patient non-compliance in global HIV management due to social, economic, behavioral, and healthcare access barriers, potentially disconnecting many PLHIV from the HIV care continuum. Therefore, to boost patient engagement in clinical care and to improve overall patient outcomes, new approaches to viral load monitoring practices need to be developed to increase access, particularly in regions of high HIV prevalence.</p><p dir="ltr">Nucleic acid amplification tests (NAATs) have emerged as potent tools for monitoring viral load, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) being recognized as the benchmark due to its sensitivity and ability for real-time quantification enabled by fluorescence signal emission. Nevertheless, RT-qPCR is burdened by drawbacks including extended processing times, high operational costs, and the requirement for specialized laboratory facilities. In this study, we propose a novel method for HIV-1 viral load monitoring by integrating reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) with real-time particle diffusometry (PD). This approach allows for the continuous monitoring of changes in the diffusion of 400 nm fluorescent particles during RT-LAMP amplification, targeting the <i>p24</i> gene region of HIV-1 RNA. This enables the real-time detection of amplification curves, achieving a detection sensitivity in water samples as low as 25 virus particles per μL within a short duration of 30 minutes. Additionally, to address challenges related to amplification inhibition in complex human specimens, we developed a power-free sample processing system specifically designed for extracting HIV-1 RNA from both whole blood and plasma.Top of FormBottom of FormThis system modifies a commercially available spin-column protocol by integrating a syringe device and handheld bulb dryer, thus eliminating the requirement for a centrifuge. The adaptation allows for the completion of the entire extraction procedure, encompassing viral lysis, RNA capture, washing, and elution of purified HIV-1 RNA, within a timeframe of less than 16 minutes. Subsequent analyses, including RT-LAMP and RT-qPCR, demonstrate a limit of detection of 100 copies per μL and an average RNA recovery of 32% (for blood) and 70% (for plasma) in the elution fraction. Further investigations emphasize the significant presence of purified RNA in the spin column volume (termed as dead volume), and the cumulative recovered RNA copies align with those obtained using the gold standard centrifugation extraction method. Ultimately, we incorporated the real-time quantitative PD-RT-LAMP assay onto a field-compatible handheld portable platform suitable for field use, featuring built-in quality control measures. This platform enables sample-to-answer viral load testing near the point of care (POC). Subsequently, we undertook essential preparatory steps, such as reagent drying to obviate the need for cold storage, initial device calibration, and hands-on training of laboratory personnel regarding device operation, to validate device performance within a cohort of individuals living with HIV (PLHIV). These innovations facilitate quick and comprehensive viral load determination, offering promise for enhanced HIV management and patient care</p>

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Introdução: A meningite tuberculosa (MTB) é a forma mais grave e fatal de tuberculose. O diagnóstico oportuno e o tratamento adequado e precoce são os principais fatores associados com o bom prognóstico. Os métodos utilizados na prática médica diária - achados clínicos, exames de imagem e análise de líquido cefalorraquidiano (LCR) - têm baixa acurácia. A pesquisa do DNA do Mycobacterium tuberculosis no LCR através da reação em cadeia da polimerase (PCR, do inglês polimerase chain reaction) com a metodologia nested é promissora, especialmente quando associada à praticidade da amplificação do DNA em tempo real. Objetivo: Avaliar o valor diagnóstico da nested PCR em tempo real (nRT-PCR, do inglês nested real-time PCR) na investigação de pacientes com MTB. Métodos: Estudo observacional realizado em duas fases: uma prospectiva e outra retrospectiva. Na fase prospectiva, foram incluídos pacientes com suspeita de MTB internados no Instituto de Infectologia Emílio Ribas (IIER). Informações clínicas, laboratoriais e radiológicas foram coletadas, assim como amostra de LCR de todos os pacientes. A partir de critérios internacionais padronizados, os pacientes foram categorizados como \"MTB Definitiva\", \"MTB Provável\", \"MTB Possível\" e \"Não MTB\". A nRT-PCR, utilizando o gene alvo mpt64, foi realizada em todas as amostras de LCR no Laboratório de Meningites Bacterianas do Instituto Adolfo Lutz. Sensibilidade, especificidade e intervalos de confiança (IC 95%) da nRT-PCR foram calculados com base no padrão-ouro (cultura positiva para M. tuberculosis ou isolamento de BAAR no sistema nervoso central) e nos pacientes com outros diagnósticos estabelecidos (Não MTB). Também foi calculada a proporção de pacientes com a nRT-PCR positiva em cada categoria clínica. Na fase retrospectiva, foi realizada uma revisão de prontuários de pacientes que tiveram a nRT-PCR solicitada no IIER e no Centro de Referência e Treinamento em DST/AIDS. Os mesmos procedimentos de categorização diagnóstica, cálculos de sensibilidade e especificidade foram adotados. Resultados: Na fase prospectiva, foram incluídos 102 pacientes, sendo 92 deles infectados por HIV. Nove deles tiveram o padrão-ouro positivo e foram classificados como \"MTB Definitiva\" e 81 deles tiveram outros diagnósticos estabelecidos (\"Não MTB\"). A sensibilidade e a especificidade da nRT-PCR foi 100% (IC95%:70-100 e 95-100, respectivamente). A positividade da nRT-PCR na categoria \"MTB Provável\" foi 50% (4/8 pacientes) e 25% na \"MTB Possível\" (1/4). Na fase retrospectiva, 56 pacientes foram incluídos, sendo 48 infectados por HIV. A nRT-PCR teve sensibilidade de 83% (5/6) e especificidade de 100% (0/45). A positividade na categoria \"MTB Provável\" foi 60% (3/5) e não houve pacientes classificados como \"MTB Possível\". Conclusão: A nRT-PCR apresentou boa sensibilidade e ótima especificidade, demonstrando seu valor diagnóstico na identificação oportuna de casos de MTB / Background: Tuberculous meningitis (TBM) is the most serious and lethal presentation of tuberculosis. Timely diagnosis and appropriated treatment are the main factors associated with good outcome. Methods used in the daily medical practice - clinical, radiological and cerebrospinal fluid (CSF) findings - have low accuracy. Search for Mycobacterium tuberculosis DNA in the CSF by polymerase chain reaction (PCR) using the nested methodology is promising, especially when combined with the practical approach of the real time DNA amplification. Objective: To evaluate the diagnostic value of a nested real-time PCR (nRT-PCR) in the investigation of patients with TBM. Methods: A two-phase observational study was carried out: prospective and retrospective. In the prospective phase, patients with suspected TBM hospitalized at \"Instituto de Infectologia Emílio Ribas\" (IIER) were included. Clinical, laboratory and radiological data were collected, as well as CSF samples of all patients. According to international standard criteria, patients were categorized as \"TBM Definite\", \"TBM Probable\", \"TBM Possible\" and \"Not TBM\". The nRT-PCR, using the mpt64 gene, was performed on all CSF sample in the Laboratory of Bacterial Meningitis, Adolfo Lutz Institute. Sensitivity, specificity and confidence intervals (95% CI) of the nRT-PCR were calculated based on the gold standard (culture positive for M. tuberculosis or AFB isolation on the central nervous system) and on patients with other established diagnoses (\"Not TBM\"). The proportion of patients with a positive nRT-PCR in each clinical category was also calculated. In the retrospective phase, medical chart review was performed in those patients who had the nRT-PCR requested in IIER and in the \"Centro de Referência e Treinamento em DST/AIDS\". The same diagnostic categorization and calculations of sensitivity and specificity were adopted. Results: 102 patients were included in the prospective phase, 92 of them HIV-infected. Nine of them had the gold standard positive and were classified as \"TBM Definite\" and 81 of them had other diagnoses established (\"Not TBM\"). The sensitivity and specificity of the nRT-PCR were 100% (95%CI: 70-100 and 95-100, respectively). The nRT-PCR positivity in category \"TBM Probable\" was 50% (4/8 patients) and 25% in \"TBM Possible\" (1/4). In retrospective phase, the nRT-PCR had a sensitivity of 83% (5/6) and specificity of 100% (0/45), among the 56 included patients (48 of them HIV infected). Positivity in \"TBM Probable\" category was 60% (3/5) and no patients were classified as \"TBM Possible\". Conclusion: The nRT-PCR showed good sensitivity and excellent specificity, showing its diagnostic value in the timely identification of TBM