Comparative Analysis of HPV Detection Efficiency: Evaluating Cobas 8800 Performance in Vaginal Self-Sampling versus Clinician-Collected Samples at a Regional Thai Hospital.

BACKGROUND: This study, conducted at a regional Thai hospital, assesses the comparative efficacy of self-collected versus clinician-collected samples for HPV detection using the Cobas 8800 system among Thai women aged 30-60. METHODS: Our methodology involved analyzing 1541 self-collected and 1398 clinician-collected samples. RESULTS: The results show a statistically significant mean difference in cycle threshold (Ct) values favoring clinician-collected samples (1.53; 95% CI: 1.18-1.87, p < 0.0001). This pattern was consistent across various age groups, with the most pronounced differences noted in the oldest cohort (50-59 years), suggesting higher detection efficiency in clinician-collected samples. The study further explored the correlation of Ct values with cytological and histological outcomes, where clinician-collected samples demonstrated superior diagnostic performance, particularly in identifying LSIL and HSIL conditions, evidenced by AUC values of 0.793 and 0.866, respectively. While self-sampling remains a viable method, with sensitivity reaching up to 48.84% for LSIL and 46.15% for HSIL, clinician collection proved more accurate, likely influencing future national screening policies. CONCLUSIONS: This work underscores the need for robust sample collection methods and the importance of ongoing enhancements to self-sampling assays and techniques to ensure their efficacy in cervical cancer screening programs.

Dual approaches in pharmacogenetics: Developing PCR-SSP and RT-PCR methods for HLA-B*13:01 screening to prevent dapsone and Co-trimoxazole SCARs.

Dapsone and co-trimoxazole are potent antibiotics for treating various infections and inflammations. However, several studies reported the strongly association between severe cutaneous adverse drug reactions (SCARs) to both drugs and the HLA-B*13:01 allele. Rapid and reliable screening for the HLA-B*13:01 allele can mitigate the risk of dapsone-induced SCARs. We developed two methods, multiplex sequence-specific primer PCR (PCR-SSP) and real-time PCR (RT-PCR), tailored for different clinical settings. These methods were optimized to minimize false positives among the Thai population. Clinical validation demonstrated excellent reproducibility, with both methods showing 100 % concordance in repeated tests. PCR-SSP achieved a limit of detection as low as 100 pg of genomic DNA, while RT-PCR reached 1 pg. Overall statistical accuracy was 100.00 % (95 % CI: 98.18 %-100.00 %). Screening for drug-related HLA alleles is crucial for reducing mortality from severe cutaneous adverse drug reactions, especially dapsone hypersensitivity syndrome (DHS) and dapsone-induced hypersensitivity reactions (DIHRs). Our screening approach for dapsone can also be extended to co-trimoxazole, representing a significant advancement in personalized medicine and preemptive pharmacogenetic testing for tailored patient care and safety, albeit further validation in diverse ethnic populations is warranted to ensure universal applicability.

Evaluation of rapid diagnostic test kits for detection of Treponema pallidum antibody.

Rapid syphilis testing plays a crucial role in global health strategies, addressing the urgent need for prompt and accurate diagnostics, especially in settings with limited resources. Despite their practical utility, these tests often lack thorough validation, leading to concerns about their efficacy and reliability. This study aims to evaluate two prototypes of the Onsite Syphilis Ab Combo Rapid Test (Fd and Ff) and compare their performance with the established chemiluminescent microparticle immunoassay (CMIA) method. Employing a reverse algorithm approach, the study analyzed 450 serum samples, including those from syphilis patients, healthy individuals, and cases with potential cross-reactions. Results of the rapid test kit were then correlated with CMIA findings, RPR, and TPPA titers. The results showed that prototype Fd exhibited a sensitivity of 100.0%, specificity of 98.8%, positive predictive value (PPV) of 8.4%, negative predictive value (NPV) of 100.00% and accuracy of 98.8%. Similarly, prototype Ff exhibited sensitivity of 100.0%, but with a slightly higher specificity of 99.6%, PPV of 21.5%, NPV of 100.0% and accuracy of 99.6%. Moreover, both prototypes Fd and Ff of the Onsite Syphilis Ab Combo Rapid Test demonstrated significant efficacy diagnostic tool, offering clear and straightforward interpretation for clinicians in varied CMIA, RPR and TPPA titer scenarios. The Onsite Syphilis Ab Combo Rapid Test prototypes, Fd and Ff, demonstrated high sensitivity and specificity, comparable to CMIA methods. The effectiveness highlights their suitability for syphilis screening, particularly in non-laboratory settings or situations requiring immediate results. The validation of these prototypes supports their integration into current syphilis diagnostic algorithms, potentially contributing to improved public health outcomes.

Establishing age and gender-specific serum creatinine reference ranges for Thai pediatric population.

Accurate assessment of kidney function in children requires age and gender-specific reference ranges for serum creatinine. Traditional reference values, often derived from adult populations and different ethnic backgrounds, may not be suitable for children. This study aims to establish specific reference ranges for serum creatinine in the Thai pediatric population, addressing the gap in localized and age-appropriate diagnostic criteria. This retrospective study analyzed serum creatinine levels from Thai children aged newborn to 18 years, collected from the Laboratory Information System of the Queen Sirikit National Institute of Child Health from January 2017 to December 2021. The Bhattacharya method was employed to establish reference ranges, considering different age groups and genders. The study compared these newly established reference values with international studies, including those of Schlebusch H., Pottel H., and Chuang GT., to validate their relevance and accuracy. A total of 27,642 data entries (15,396 males and 12,246 females) were analyzed. The study established distinct reference ranges for serum creatinine, which varied significantly across different age groups and between genders. These ranges were found to gradually increase with age from 2 months to 18 years. The study also highlighted notable differences in reference values when compared with other ethnic populations. The study successfully establishes tailored reference ranges for serum creatinine in Thai children, providing a valuable tool for more accurate diagnosis and monitoring of kidney health in this demographic. This initiative marks a significant advancement in pediatric nephrology in Thailand and suggests a need for continuous refinement of these ranges and further research in this area.

DROP and READ: a paper-based device combined with portable readout for ABO, Rh (D, C, c, E, e) and Mia phenotyping.

The transfusion of ABO-compatible blood with blood with an unknown phenotype may result in alloimmunization, particularly in multitransfused patients. Minor blood-group phenotyping and the selection of negative blood for specific antigens reduce post-transfusion complications. With this study, a device called the DROP and READ instrument was developed with a PAD (paper-based device) and various software to phenotype ABO, Rh (D, C, c, E, e), and Mia antigens. EDTA (Ethylene diamine tetra-acetic acid) blood samples were collected from donors, volunteers, and newborns, and were then tested with the DROP and READ instrument according to the lateral flow and RBC agglutination principle. The results were compared with those obtained by using a routine column agglutination test or the tube method. Results: a total of 205 samples were tested (150 from EDTA blood donors, 50 from EDTA blood volunteers, and 5 from the cord blood of newborns). The device yielded 100% accuracy, sensitivity, and specificity, a positive predictive value, and a negative predictive value when interpreting the ABO, Rh (D, C, c, E, e), and Mia antigens. The DROP and READ instrument is developed to automatically interpret the results, and the device provided endpoint results without centrifugation and prevented misinterpretations from human error.

Phenotyping of minor blood groups (C, c, E, e, and Mia) using a paper-based device and image-based high-throughput detection.

Testing ABO and D (Rh) are priorities before blood transfusion, and the minor blood group antigen should be matched to reduce alloimmunization. In a recent study, a paper-based device (PAD) was developed for C, E, c, e, Mia phenotyping, combined with image-based high-throughput detection. A total of 148 ethylenediamine tetra acetic acid (EDTA) blood samples were used to evaluate and create an optimal criterion using OpenCV for high-throughput interpretation. Results revealed that anti-C, -c, -E, -e, and -Mia were successful for blood group phenotyping with area under the receiver operating characteristics curve (AUC) of 1.000 (95% confidence interval [CI], 0.976-1.000), 0.984 (95% CI, 0.984-0.997), 0.997 (95% CI, 0.970-1.000), 0.994 (95% CI, 0.965-1.000), and 1.000 (95% CI, 0.976-1.000), respectively. The validation of these systems for blind blood samples (n = 56) showed 100% sensitivity, specificity, and accuracy compared with the gel card method. PAD with image-based interpretation can be used as an alternative to minor blood group phenotyping without the need for electric power equipment and well-trained personnel. Moreover, this proposed method would help build phenotype databases of blood donors or patients for the preparation of panel cells, find antigen-negative compatible blood for patients with multiple alloantibodies, and prevent alloimmunization in multitransfused patients.

Development of Mia Phenotyping Using Paper-Based Device.

The MNS7 (Mia) blood group antigen is found at a different prevalence among different ethnic groups. Anti-Mia can cause hemolytic disease of the fetus and newborn (HDFN) and both acute- and delayed-type hemolytic transfusion reactions (HTR). Mia typing should be performed in donors to prevent life-threatening hemolytic transfusion reactions. The gel card and standard tube methods still need specialized equipment, centrifugation, and expertise for result interpretation. We used a novel paper-based analytical device (PAD) pre-coated with monoclonal IgM anti-Mia for Mia phenotyping. We measured grey pixel intensity in blood typing results for interpretation processing using OpenCV at the sample (SP) and elution parts (EP); furthermore, we used the SP: EP ratio and F-score as analysis criteria. We typed 214 blood EDTA samples with PAD-Mia and then compared with gel card results for setting an analysis criterion. We observed 100% sensitivity, specificity, and accuracy when we applied the SP: EP ratio and F-score with the optimal criterion (1.07 and 0.17 for SP: EP ratio and F-score, respectively). The validation of PAD-Mia typing for blood donor samples (n = 150) via F-score gave 100% sensitivity and specificity when compared with the gel card method; therefore, we argue that PAD-Mia typing can be used for Mia phenotyping without sero-centrifugation. Moreover, to study the correlation between genotype and phenotype, PCR-SSP was performed to identify GYP(B-A-B) hybrids. The results revealed that all Mia+ blood samples gave a positive with GP. Hut, GP. HF, GP. Mur, GP. Hop, and GP. Bun. Results of the gel card method and PCR-SSP were concordant. Hence, using PAD-Mia typing in blood donors would be helpful for creating a phenotype database of blood donors for reducing alloimmunization risks.

Evaluation of the ESR fast detector and Improve® ESR analyzer as modified Westergren methods for erythrocyte sedimentation rate.

The erythrocyte sedimentation rate (ESR) has been commonly ordered in hematology laboratories and used to screen for monitoring responses to therapy and identifying inflammatory conditions. To overcome the limitations of traditional ESR measurements, various methods have been developed and compared to the established reference method. This study evaluates the analytical performance of ESR fast detector and Improve® ESR analyzer compared to the reference method. Method validation and comparison were performed in 189 volunteer blood samples according to the International Council for Standardization in Hematology recommendations. The analytical efficacy of ESR fast detector and Improve® ESR analyzer was also assessed and compared with the reference method and C-reactive protein (CRP) levels. The results demonstrated that the precision of ESR fast detector and Improve® ESR analyzer was considered as the acceptance criterion for the ESR measurement. The method comparison analysis between the two modified Westergren methods and reference method demonstrated a strong correlation with the Spearman's rank correlation coefficient of 0.94, with a mean difference of -2.1 and -7.7 mm/h in the ESR fast detector and Improve® ESR analyzer, respectively. Analysis of the area under the receiver operating curve illustrated a high analytical performance compared to the reference method and CRP level. The measurement of ESR level using the ESR fast detector and Improve® ESR analyzer is a reliable method and has a high analytical performance, which can be used instead of the reference method for screening inflammatory conditions.

Development of label-free electrochemical impedance spectroscopy for the detection of HLA-B*15:02 and HLA-B*15:21 for the prevention of carbamazepine-induced Stevens-Johnson syndrome.

BACKGROUND: Carbamazepine (CBZ) is an FDA-approved anticonvulsant that is widely used to treat epilepsy, bipolar disorder, trigeminal neuralgia and chronic pain. Several studies have reported a strong association between HLA-B*15:02 and carbamazepine-induced Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). However, the HLA-B75 serotype (HLA-B*15:02, HLA-B*15:08, HLA-B*15:11 and HLA-B*15:21) has been found in patients with carbamazepine-induced SJS/TEN. METHODS: This study aimed to develop label-free electrochemical impedance spectroscopy (EIS) for the detection of HLA-B*15:02 and HLA-B*15:21 after PCR-SSP amplification. A total of 208 DNA samples were tested. The impedance was measured and compared to standard gel electrophoresis. RESULTS: The developed label-free EIS identified HLA-B*15:02 and HLA-B*15:21 alleles with 100% sensitivity (95% CI: 86.773%-100.000%) and 95.05% specificity (95% CI: 90.821%-97.714%), comparable to commercial DMSc 15:02 detection kits. CONCLUSIONS: We successfully developed a novel PCR-SSP associated with signal impedance changes to detect the HLA-B*15:02 allele and HLA-B*15:21 without downstream amplicon size analysis that is suitable for screening individuals before indication of CBZ therapy.

Portable paper-based device for ABO and RhD typing using smartphone interpretation: Optical answer sheet reading concept.

The slide method for ABO blood group typing is commonly used for mobile blood donation and field applications because the test is easy, cost-effective, can be completed in a few minutes and requires a small volume of reagents. However, the reaction must be observed by an individual with expertise within 2 min; otherwise, drying of the reagent will give a false positive result. Moreover, the blood typing reagents must be stored at 4 °C. The present study aimed to create a paper-based device for ABO and RhD blood typing and combine an optical answer sheet reading concept to read and interpret the results with Android smartphones. The invention of this device involved the use of simple filter paper and conjugate pads that were treated with anti-A, -B and -D antibodies. Blood type can be visually identified from the detection zone at the end of the filter paper. An Android smart phone was designed to read the detection zone, interpret the data and subsequently report the results on the user's smartphone. A helpful color chart was also designed for blood typing interpretation by the naked eye. The use of smartphones can reduce human error in data reading and interpretation. In conclusion, ABO and RhD typing with paper-based devices using a smartphone interpretation may provide further advantages for home-based users, mobile blood donation sites and field applications.

Differential diagnosis of Plasmodium falciparum and Plasmodium vivax in mixed infection by colorimetric nanogold probes.

Malaria is an infectious disease reported mostly in the tropical region. The most severe human malaria is Plasmodium falciparum since it can cause cerebral malaria. Therefore, the presence of P. falciparum either in single or mixed infection needs accurate diagnosis. In some mixed infections, the presence of P. falciparum may be cryptic which cannot be detected by microscopic examination. The molecular diagnosis is required in these cases. Many methods based on amplification of malaria parasite genes have been developed but most of them need sophisticated instruments. Here, we created a colorimetric method using probe immobilized gold nanoparticles (AuNPs) to detect the malaria parasite gene. Color changes rely on salt-induced aggregation of AuNPs in the presence or absence of DNA hybridization. Color changes could be observed either by a naked eye or UV-vis spectrophotometer. By this approach, single infection by the most common malaria parasite, P. falciparum or P. vivax could be differentially identified. Mixed infection of these two malaria species could also be clearly diagnosed including cases of cryptic P. falciparum. The novel nanogold based molecular malaria diagnosis is sensitive, specific, rapid and cheap ($0.94). The prepared nanogold malaria probes are stable for up to 3 months indicating their filed application in remote areas.

Enhancement of bloody fingerprints on non-porous surfaces using Lac dye (Laccifer lacca).

It is important that fingermark enhancement techniques are safe and simple to carry out. Many chemicals are widely used to enhance and develop bloody fingermark. However, the use of natural products for fingermark detection and examination has several advantages and challenges. In this study, Lac dye (Laccifer lacca) was used to enhance bloody fingermarks on various types of non-porous and porous materials. Bloody fingermarks were deposited using a depletion series technique on eleven different surfaces. To assess the efficiency of Lac dye stain, comparisons were performed with Amido black stain as a reference method. Results revealed the similarity between Lac dye and Amido black on non-porous materials, in terms of both fingermark grades, and color intensity. However, Lac dye showed relatively low performance for enhancing and developing bloody fingermarks on porous materials. This indicates that Lac dye can be beneficially used as an alternative to chemicals such as Amido black on a non-porous surface. Further study into Lac dye formulation on porous materials is recommended.

Evaluation of quantitative biosensor for glucose-6-phosphate dehydrogenase activity detection.

Neonatal jaundice is a common and severe disease in premature infants with Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency. The World Health Organization (WHO) has recommended screening for G-6-PD deficiency in newborns for early recognition as well as to prevent unwanted outcomes in a timely manner. The present study aimed to assess a point-of-care, careSTARTTM G6PD biosensor as a quantitative method for the diagnosis of G-6-PD deficiency. Factors influencing the evaluation of G-6-PD enzyme activity were examined in 40 adults, including ethylenediaminetetraacetic acid (EDTA) anticoagulant, hematocrit concentration, storage temperature and time. Analytic performance of the careSTARTTM G6PD biosensor was evaluated in 216 newborns and compared with fluorescent spot test (FST) and standard quantitative G-6-PD enzyme activity (SGT) assay. The results of factors affecting the G-6-PD enzyme activity showed that the activity determined from finger-prick was not statistically different from venous blood (p = 0.152). The G-6-PD value was highly dependent on the hematocrit and rose with increasing hematocrit concentration. Its activity was stable at 4°C for 3 days. Reliability analysis between the careSTARTTM G6PD biosensor and SGT assay showed a strong correlation with a Pearson's correlation coefficient of 0.82 and perfect agreement by intraclass correlation coefficient (ICC) of 0.90. Analysis of the area under the Receiver Operating Curve (AUC) illustrated that the careSTARTTM G6PD biosensor had 100% sensitivity, 96% specificity, 73% positive predictive value (PPV), 100% negative predictive value (NPV) and 97% accuracy at 30% of residual activity. While the diagnostic ability for identifying G-6-PD deficiency had 78% sensitivity, 89% specificity, 56% positive predictive value (PPV), 96% negative predictive value (NPV) and 88% accuracy when stratified by gender. The careSTARTTM G6PD biosensor is an attractive option as a point-of-care quantitative method for G-6-PD activity detection. Quantification of G-6-PD enzyme activity in newborns is the most effective approach for the management of G-6-PD deficiency to prevent severe jaundice and acute hemolysis.

Evaluation of black glutinous rice (Oryza sativa L) extract as a novel nuclear stain for human sperm head assessment by microscopic examination.

OBJECTIVE: To compare black rice (Oryza sativa L) extract with three different staining methods for human sperm head assessment. METHODS: Semen samples were collected from 34 volunteers. Four smears of each ejaculate were prepared for staining using the rapid Papanicolaou (PAP) stain, SpermBlue, DipQuick, and black rice extract. The percentage of defective sperm heads (mean±standard deviation) was compared. RESULTS: Black glutinous rice extract, a natural dye, was used instead of hematoxylin to stain the nuclei of the sperm heads. The percentage of defective sperm heads showed a significant difference between black rice extract and DipQuick (p=0.000). In contrast, black rice extract and rapid PAP showed no statistically significant difference (p=0.974). A strong correlation (r =0.761) was found between the findings obtained using rapid PAP and black rice extract. In contrast, a weak correlation (r =0.248) was obtained between DipQuick and black rice extract for the percentage of defective sperm heads. CONCLUSION: The results showed good agreement and a strong correlation between the rapid PAP and black rice extract stains. The advantages of black rice extract as a novel substitute for hematoxylin for nuclear staining include ease of preparation, local availability, and favorable nuclear staining properties. Further studies could also focus on comparing staining techniques in clinical samples.

Evaluation of natural dyes for human spermatozoa morphology assessment.

Dye extracts from plants have been valuable not only for the economy but also environmental sustainability. There have been many reports on the utilization of natural dyes extracted from various sources for staining of biological tissues. This study aimed to investigate the extraction of natural dye from black rice (Oryza Sativa), butterfly pea (Clitoria ternatea), fresh roselle (Hibiscus sabdariffa), and mulberry (Morus alba) to stain of human spermatozoa for morphology assessment. The results showed that black rice extracted from solvents C containing 5 ml of absolute ethanol, 10 g of potassium alum and 100 ml of distilled water is the best dye for human spermatozoa evaluation comparable to the rapid PAP and Dip quick® stain. Effectiveness of the process was found with black rice extract stain by using 2 steps for 15 min. There were no statistically significant differences in the parameters of head, midpiece, tail and background for human sperm morphology assessment comparable to rapid PAP and Dip quick® stain (p > 0.05) unless the midpiece compartment when compare to rapid PAP. This finding suggests that the black rice extracted has potential for use as an alternative dye for human spermatozoa morphology evaluation. The usefulness of black rice extracts will decrease the expense for purchasing synthetic dyes and reduce their adverse effects on human and environment.

The evaluation of Oryza sativa L (Black rice) extracts for detection of spermatozoa on the clothing and vaginal swab samples.

Investigation of sexual assault cases from the evidence involving vaginal swab, clothing and others is examined by a forensic scientist. The explanation of trace findings on spermatozoa on clothing is often problematic due to the use of different staining methods. Conventional staining method used either Papanicolaou (PAP) or Dip quick® stain as synthetic dyes which are expensive imported material and harmful to human health. Therefore, the present study aims to determine the ability of Oryza sativa L (black rice) extract as a natural dye to detect spermatozoa on the clothing and vaginal swab casework samples for routine forensic examination. Results revealed that black rice extract has a highly effective for detecting spermatozoa on cloth and vaginal swab casework samples. There was no significantly different in the detection of spermatozoa compared with rapid PAP stain and Dip quick® stain. Results also showed that the staining of vaginal swab casework with black rice extracted can be used for PCR amplification of centromeric alphoid repeat gene on chromosome Y for 60 days. Moreover, the DNA extracted from stained semen slide generates a full profile of 16 alleles of STR typing. The results indicate that a new natural staining dye which extracted from black rice can be used to detect spermatozoa and identify a person from the trace evidence. The application of natural dyes for routine staining of spermatozoa from forensic specimens will decrease the expense to be spent in purchasing the synthetic dye and reduce their side effects on human and environment.

Development of Visual Detection of α-Thalassemia-1 (the - -SEA Deletion) Using pH-Sensitive Loop-Mediated Isothermal Amplification.

Detection of α-thalassemia-1 (α-thal-1) carriers provides valuable insight for genetic consulting in prevention and control programs for couples who are at risk of conceiving a fetus with severe thalassemia, both Hb Bart's hydrops fetalis and hemolytic Hb H disease. The traditional method is complicated, time-consuming and requires high instrument cost and expertise. Loop-mediated isothermal amplification (LAMP) based on pH-sensitive dye technology, shows all the characteristics required of a real-time analysis with simple operation for potential use in the clinical diagnosis of high incidence α-thal-1 [Southeast Asian (SEA) or - -SEA deletion]. Four primers specific for six distinct regions of the α-globin gene deletion were designed and analyzed by LAMP using the pH-indicator dye, phenol red. The amplification of the - -SEA deletion changed the color of phenol red from pink to orange. The diagnostic ability of detection of the - -SEA deletion by pH-sensitive LAMP was validated using both known and unknown blood samples and compared to the conventional polymerase chain reaction (PCR) method. Color inspection of pH-sensitive LAMP products could clearly identify the - -SEA deletion. There was no cross reaction with a normal α-globin gene, α-thal-1 Thai (- -THAI deletion), α-thal-2 [-α3.7 (rightward) and -α4.2 (leftward) deletion] and ß-thalassemia (ß-thal). Detection of the SEA deletion by pH-sensitive LAMP was consistent as compared to conventional PCR. The pH-sensitive LAMP method developed for this deletion carrier diagnosis has high sensitivity, specificity, simplicity, and requires simple instrumentation that makes it applicable for resource-limited laboratories in rural areas of developing countries.

Genotyping of α-thalassemias by the colorimetric nanogold probes.

BACKGROUND: The novel colorimetric nanogold probe was created to genotype subgroups of the mostly found α-thalassemias. They are α-thalassemia 1 (SEA and THAI deletion) and α-thalassemia 2 (3.7-kb and 4.2-kb deletion). METHODS: The genotyping was performed by two-steps hybridizations. First step was hybridization of target DNA with the nanogold mixed probes of either α-thalassemia 1 or α-thalassemia 2. No hybridization in both reactions showing blue color indicated absence of abnormal genes causing these α-thalassemias. Positive reaction showing either red or purple color was further analyzed in second hybridization with the nanogold single probe. Positive of α-thalassemia 1 was genotyped with the single probes of both SEA and THAI deletion while those of α-thalassemia 2 were genotyped with both 3.7-kb and 4.2-kb deletion. RESULTS: Genotypic potency of the nanogold mixed and single probes was evaluated using both known diagnosed and suspected clinical samples. The results by naked eye were consistence with those analyzed by standard agarose gel electrophoresis. CONCLUSIONS: Potency of the colorimetric nanogold α-thalassemia probes was accurate, precise, sensitive, specific, simple, cheap and field applicable. Color reaction was simply visualized by naked eye. This development is an example of colorimetric molecular diagnosis which can be applied in any genetic detection.

Silver quartz crystal microbalance for differential diagnosis of Plasmodium falciparum and Plasmodium vivax in single and mixed infection.

The most severe form of malaria is cerebral malaria caused by Plasmodium falciparum. Standard malaria diagnosis is Giemsa stained peripheral blood smear but false negative findings are always reported. Moreover, mixed infections are underestimated by routine microscopy. Many methods have been developed to overcome these disadvantages and the most specific and sensitive is molecular diagnosis. Specific malaria genes are amplified by polymerase chain reaction (PCR) and many post-PCR methods have been created. We developed a gold fabricated quartz crystal microbalance (QCM) as a post-PCR method of malaria diagnosis. In this work a cheaper silver fabricated QCM was developed to identify both single and mixed infection of P. falciparum and Plasmodium vivax. The biotinylated malaria probe was immobilized on silver surface via specific avidin-biotin interaction. The target DNA fragment of 18s rRNA gene was amplified and hybridized with a QCM immobilized probe. Mass changes due to DNA hybridization were indicated by changes of quartz resonance frequencies. Validation showed that malaria silver QCM had high diagnostic potency. Evaluation of suspected 67 febrile blood samples from malaria endemic area demonstrated that the malaria silver QCM could identify both false negative and misdiagnosis cases of routine microscopy. The analysis cost of malaria silver QCM was $1/sample and analysis time was 30 min after blood collection. The malaria silver QCM is stable at tropical temperature for up to 6 months. Thus, it can be transported to be used in a remote endemic area. Thus, the malaria silver QCM is accurate, precise, rapid, cheap, and field applicable.

Molecular diagnosis of α-thalassemias by the colorimetric nanogold.

A new application of gold nanoparticles (AuNPs) as a colorimetric method for gene detection of α-thalassemia 1 (SEA deletion) is reported here for the first time. This technique is based on color changes from salt-induced aggregation of un-hybridized nanogold probes after hybridization with the target DNA. Specific DNA probes were synthesized, thiol modified and conjugated on the surface of AuNPs. The target DNA was amplified and hybridized with the AuNPs-immobilized probe. Salt solution (NaCl) was added to induce aggregation of the un-hybridized nanogold probes. The color changes were visualized either by the naked eye or by UV-vis spectrophotometry at 520 nm. By this nanogold colorimetric method samples carrying normal α-globin genes could be successfully identified from samples carrying α-globin genes causing α-thalassemia 1 (SEA deletion), either as a carrier or disease form. Results demonstrated that the new colorimetric nanogold method is a definite gene diagnosis of α-thalassemia. It is accurate, simple, rapid, specific, sensitive, and cost effective. It is also a promising point-of-care testing (POCT) method for thalassemias and other genetic disorders. The new colorimetric nanogold is a method of choice for areas where access to sophisticated molecular diagnosis is limited.