CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus.

Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.

A Comparative Evaluation of HbA1c Measurement Methods and Their Implications for Diabetes Management.

In this study, we assessed the correlations between hemoglobin A1c (HbA1c) measurements obtained using three different diagnostic methods, namely reversed-phase cation-exchange chromatography, high-performance liquid chromatography, and lateral flow immunoassay (LIFA) with an AnyLab F instrument. HbA1c levels measured with the AnyLab F instrument and those measured with the HA8190V, HA8180, and D100 instruments were strongly correlated. High R-square values and low p-values indicated significant and reliable correlations, supporting the clinical interchangeability of these methods. Notably, demographic and clinical analyses revealed uniform HbA1c levels across age groups, suggesting minimal age-related variations in HbA1c levels in the cohort. This finding has implications for diabetes management strategies across different age groups, emphasizing the versatility of the AnyLab F instrument. Overall an average HbA1c level of 7.857% among diabetes mellitus-diagnosed participants suggests moderately elevated HbA1c levels, underscoring the need for improved diabetes management. Younger individuals exhibited lower HbA1c levels, potentially owing to heightened awareness and treatment plan adherence. Conversely, older adults had higher HbA1c levels, likely influenced by age-related changes and comorbidities. Larger sample sizes and a comprehensive evaluation of various measurement principles are needed to strengthen the findings herein. Additionally, exploring additional biomarkers and assessing LIFA performance in larger sample sets will advance the clinical utility of HbA1c measurements.

Development of a Rapid Diagnostic Kit for Congestive Heart Failure Using Recombinant NT-proBNP Antigen.

Background and Objectives: In patients with congestive heart failure, brain natriuretic peptide (BNP) and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) are released due to excessive heart muscle expansion; they can be used for the early detection, progress monitoring, and treatment of congestive heart failure. Recently, considerable efforts have been made to develop an NT-proBNP-based biomarker for detecting heart failure. This study attempts to develop a rapid and accurate congestive heart failure diagnostic kit using NT-proBNP. Materials and Methods: A new gene based on NT-proBNP was selected, recombined, and expressed in Escherichia coli strains, and then monoclonal antibodies were produced using the hybridoma technique. Additionally, antigen-antibody reactivity was confirmed using indirect enzyme-linked immunosorbent assay (ELISA). Furthermore, the first pair and full-strip pair tests were conducted to select candidate clones; these were applied to a rapid diagnosis kit based on gold conjugates and compared with other currently available antigens. Results: NT-proBNP-based antigens with high specificity and monoclonal antibodies were produced, and the optimal antigen-antibody reactivity was confirmed using indirect ELISA. The first pair and full-strip pair tests were performed to select the optimal candidate clones, and a rapid diagnosis kit with excellent reactivity was developed by applying these to a rapid diagnosis kit based on gold conjugates. Conclusions: The development of this rapid diagnosis kit with excellent performance in congestive heart failure is expected to improve disease management by providing an early assessment of the risk of heart failure.

Evaluation of commercially available three dengue rapid diagnostic test kits for diagnosis of acute dengue virus infection at the point-of-care setting in Myanmar.

Early and accurate diagnosis of dengue virus (DENV) infection is very important and Rapid Diagnostic Test (RDT) Kits are been used as a point-of-care test to check DENV infection. A Hospital and Laboratory-based descriptive study was conducted at 550-bedded Mandalay Children Hospital in 2018. Acute-phase serum samples were collected from 202 dengue suspected patients to evaluate the efficacy of RDT Kits for the diagnosis of DENV infection. Commercially available three test kits which include: ((i) CareUs Dengue Combo, Korea, (ii) Humasis Dengue Combo, Korea and (iii) Wondfo Dengue Combo, China) were validated against WHO-based reference standard tests. 140/202 patients (69.3%) was confirmed to have DENV infection. All four serotypes of dengue viruses (57 DENV-1, 7 DENV-2, 6 DENV-3 and 10 DENV-4) were identified from 80 dengue confirmed patients and DENV-1 was the dominant serotype. Combining the NS-1 antigen and IgM antibody results from the CareUs Dengue Combo Kit gave the best sensitivity (92.1%, 95% CI 86.4%-96.0%) and specificity (75.8%, 95%CI 63.3%-85.8%). Among the three RDT Kits, the performance of CareUS Kit was better than the other two. This study explored the evidence of the usefulness of RDT Kits at the point-of-care setting for diagnosis of acute dengue infection.

Evaluation of first rapid diagnostic kit for Anti-Crimean-Congo Hemorrhagic Fever virus IgM antibody using clinical samples from Iran.

Crimean-Congo Hemorrhagic Fever (CCHF) is a potentially fatal tick-borne viral disease, which is in the WHO list for emerging infections likely to cause major epidemics in the near future. Early diagnosis of CCHF is very important for both patient treatment and infection control. An efficient CCHF rapid test therefore is of great significance. This study was conducted to evaluate the sensitivity and specificity of the first CCHF rapid diagnostic kit (CCHF Sero K-SeT, CORIS BioConcept, Belgium) for detection of IgM specific antibody in patients' sera or plasma, using 87 clinical serum samples from Iranian patients. Although the assay showed an acceptable specificity of 92.9% (13/14), a low sensitivity rate of 39.7% (29/73) was observed. There was no association between the results of CCHF rapid diagnostic kit and the genotype of CCHF virus. This evaluation revealed that the CCHF Sero K-SeT is not suitable for screening of CCHF suspected cases due to its poor sensitivity.

Diagnostic accuracy of rapid diagnostic tests for the early detection of leptospirosis.

BACKGROUND: Leptospirosis is often misdiagnosed with several other tropical febrile illnesses in Malaysia due to similarities in clinical manifestations. Although treatment regimens could be started based on clinical judgments, early diagnosis has become paramount as a guide to chemotherapeutic interventions. Confirmed laboratory diagnosis through MAT or PCR is time consuming and usually available only in reference laboratories and not practical in healthcare settings. Rapid and easy to perform diagnostic tests are widely used in these settings as the point of care diagnosis. The present study was undertaken to compare the diagnostic performance of two IgM based immunodiagnostic assay kits for acute leptospirosis. METHODS: A total of 50 serum samples were collected from patients clinically suspected for acute leptospirosis on admission in the Hospital Serdang, from June 2016 to June 2017. All the samples were subjected to MAT, lipL32 PCR and the two rapid tests (Leptocheck-WB and ImmuneMed Leptospira IgM Duo Rapid test). RESULTS: Out of the 50 clinically suspected patients sampled, 19 were confirmed positive for leptospirosis. Six (12%) were confirmed by MAT and 13 (26%) by PCR. Similarly, of the 50 clinically suspected cases, 17 (34%) showed positivity for Leptocheck-WB and 7 (14%) for ImmuneMed Leptospira IgM Duo Rapid test. The overall sensitivity and specificity was 47.37% and 80.65% for Leptocheck-WB, and 21.05% and 90.32% for ImmuneMed Leptospira IgM Duo Rapid test. In another set of previously confirmed MAT positive samples (1:400-1:3600) obtained from a reference laboratory, Leptocheck-WB showed higher sensitivity (90.72%) than ImmuneMed Leptospira IgM Duo Rapid test (40.21%), and comparable specificity for ImmuneMed Leptospira IgM Duo Rapid test (88.89%) and Leptocheck-WB (82.86%). CONCLUSION: The sensitivity was higher for Leptocheck-WB and had a comparable specificity with ImmuneMed Leptospira IgM Duo Rapid test. Therefore, based on the present study, Leptocheck-WB is found to be a more sensitive rapid immunodiagnostic test for acute leptospirosis screening in hospital settings.

Development of an Influenza Rapid Diagnostic Kit Specific for the H7 Subtype.

Since the spring of 2013, human infections with H7N9 viruses have been detected in China. Some of these viruses have become highly pathogenic. Highly and low pathogenic avian influenza H7N9 viruses are currently co-circulating with the seasonal influenza A viruses H3N2 and H1N1pdm09. Prompt identification and isolation of H7N9 patients is one measure to prevent the spread of H7N9 virus and help prevent a pandemic. The majority of commercially available point-of-care rapid influenza diagnostic kits can differentiate between influenza A and B viruses, but cannot distinguish between H7N9 viruses and seasonal influenza A viruses. Accordingly, we have developed a rapid diagnostic kit specific for the H7 subtype that is accessible, easy to use. Although the detection limit of this H7 kit is one-tenth lower than that of a commercially available rapid influenza A and B diagnostic kit of similar design, except for the specificity of the monoclonal antibodies used, this kit is highly specific, detecting only H7-subtype influenza viruses, including the recent highly pathogenic H7N9 viruses from humans, and does not show any non-specific reactions with other HA subtypes. This H7 kit will be of value for the early detection of H7N9-infected patients.

Impact of Highland Topography Changes on Exposure to Malaria Vectors and Immunity in Western Kenya.

BACKGROUND: It is almost an axiom that in the African highlands (above 1,500 m) transmission of Plasmodium falciparum is limited primarily by low ambient temperature and that small changes in temperature could result in temporary favorable conditions for unstable transmission within populations that have acquired little functional immunity. The pattern of malaria transmission in the highland plateau ecosystems is less distinct due to the flat topography and diffuse hydrology resulting from numerous streams. The non-homogeneous distribution of larval breeding habitats in east African highlands obviously affects Anopheles spatial distribution which, consequently, leads to heterogeneous human exposure to malaria. Another delicate parameter in the fragile transmission risk of malaria in the highlands is the rapid loss of primary forest due to subsistence agriculture. The implication of this change in land cover on malaria transmission is that deforestation can lead to changes in microclimate of both adult and larval habitats hence increase larvae survival, population density, and gametocytes development in adult mosquitoes. Deforestation has been documented to enhancing vectorial capacity of Anopheles gambiae by nearly 100% compared to forested areas. METHOD: The study was conducted in five different ecosystems in the western Kenya highlands, two U-shaped valleys (Iguhu, Emutete), two V-shaped valleys (Marani, Fort Ternan), and one plateau (Shikondi) for 16 months among 6- to 15-year-old children. Exposure to malaria was tested using circumsporozoite protein (CSP) and merozoite surface protein immunochromatographic antibody tests. Malaria parasite was examined using different tools, which include microscopy based on blood smears, rapid diagnostic test based on HRP 2 proteins, and serology based on human immune response to parasite and vector antigens have been also examined in the highlands in comparison with different topographical systems of western Kenya. RESULTS: The results suggested that changes in the topography had implication on transmission in highlands of western Kenya and appropriate diagnosis, treatment, and control tool needed to be considered accordingly. Both plateau and U-shaped valley found to have higher parasite density than V-shaped valley. People in V-valley were less immune than in plateau and U-valley residents. CONCLUSION: Topography diversity in western Kenya highlands has a significant impact on exposure rates of human to malaria vectors and parasite. The residents of V-shaped valleys are at risk of having explosive malaria outbreaks during hyper-transmission periods due to low exposure to malaria parasite; hence, they have low immune response to malaria, while the U-shaped valleys have stable malaria transmission, therefore, the human population has developed immunity to malaria due to continuous exposure to malaria.

[Evaluation of Wondfo Rapid Diagnostic Kit for detecting Plasmodium ovale and analysis of influencing factors].

OBJECTIVE: To evaluate the Wondfo Rapid Diagnostic Kit (Pf-LDH/Pan -pLDH) for detecting Plasmodium ovale and analyze the influence of parasitaemia, concentration and polymorphism of pLDH on the performances. METHODS: A total of 100 blood samples from P. ovale patients confirmed by PCR were detected with the Wondfo Rapid Diagnostic Kit according to the manufacturers'instructions. The parasitaemia was determined by the microscopic examination. The concentration of pLDH was measured by ELISA tests. The LDH gene of P. ovale was amplified by PCR and sequenced. The influence of these three factors on the positive rate was analyzed. RESULTS: The overall positive rate of Wondfo Rapid Diagnostic Kit was 70.0% (70/100). The positive rate was 27.3% for the samples with parasitaemia ≤ 500 parasites/µl and reached 75.0%-75.4% when parasitaemia > 500 parasites/µl. The positive rate was 6.7% for samples with a low pLDH concentration (A values ≤ 0.100) and reached 95.1%-100% at a high pLDH concentration (A values > 0.100). The results of sequence analysis indicated that all the samples could be divided into 2 types, P. o. curtisi and P. o. wallikeri. The gene homology of LDH between 2 types was 97%. There were 24 single nucleotide polymorphism (s) (SNPs) between 2 types, while only 3 SNPs were non-synonymous mutations. The homology of LDH amino acid sequences between 2 types was 99%; only 3 amino acids were different. The positive rates for P. o. curtisi and P. o. wallikeri were 73.1% (38/52) and 66.7% (32/48) respectively; there was no statistically significant difference (P > 0.05). CONCLUSIONS: The Wondfo Rapid Diagnostic Kit (Pf-LDH/Pan-pLDH) performs better than most of the similar products for the detection of P. ovale, and the positive rates are closely related to the parasitaemia and concentration of pLDH, while no related to the polymorphism of pLDH gene.

Development of a Diagnostic Kit to Detect Cryptosporidium parvum and Giardia lamblia.

OBJECTIVES: This study aims to develop a high-sensitivity antibody diagnostic kit that will enable a rapid and accurate detection of Cryptospofidium parvum and Giardia lamblia in patients with diarrhea. METHODS: The cultivated C. parvum oocysts and G. lamblia cysts in each calf and dog were injected to mice to obtain antibodies, which were titrated. Spleen cells of the immunized mouse were separated and blended with myelomas to produce hybrid cell lines that form monoclonal antibodies. Using ELISA method, antibodies that specifically respond to C. parvum and G.lamblia were then selected. The cells were injected into the abdominal cavity of a BALB/c mouse to isolate hydrops abdominis containing high level of antibodies. The IgG antibody was purified using protein G gel. RESULTS: The detection limit of monoclonal antibodies for Cryptosporidium parvum and Giardia lamblia was 125 oocysts/mL and 1250 cysts/mL, respectively. In addition, during testing they did not show cross-reactivity to viruses (n = 15), bacteria (n =17), and parasites (n = 9). CONCLUSION: The rapid diagnostic antibody kit developed in this study, which specifically responds to C. parvum and G. lamblia, will be useful in detecting and monitoring diarrheal infections.