Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification.

A method for the rapid diagnosis of early dengue virus (DENV) infection is highly needed. Here, a prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed. The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. Hence, it is possible to use the RT-RPA assay in a laboratory to complement routine serology testing for dengue.

Closing the antimicrobial stewardship gap - a call for LMICs to embrace the global antimicrobial stewardship accreditation scheme.

The escalating challenge of antimicrobial resistance (AMR) poses a considerable concern for global health, particularly impacting low- and middle-income countries (LMICs). This article highlights the critical importance of tackling AMR in LMICs by adopting the Global Antimicrobial Stewardship Accreditation Scheme (GAMSAS). GAMSAS is portrayed as a holistic and sustainable strategy for antimicrobial stewardship, extending beyond accreditation to include educational programs, capacity enhancement, improved surveillance, and support for AMS policy research. While acknowledging the global uptake of the scheme, the article highlights its preliminary phase of adoption in LMICs, particularly in high-AMR burden regions like Sub-Saharan Africa. The piece stresses the imperative for LMICs to integrate GAMSAS, underscoring its significance in optimizing antimicrobial usage and patient health outcomes. It advocates for an all-encompassing approach that leverages international cooperation and sustained financial backing, crucial for the effective deployment and enduring success of antimicrobial stewardship efforts in these key areas.

Vaccine hesitancy under the lens: Nigeria's struggle against the worst diphtheria outbreak in decades.

The resurgence of diphtheria in Nigeria, culminating in an outbreak surpassing previous records, has spotlighted the critical imperative for robust immunization policies amidst a milieu of vaccine hesitancy. This commentary delineates the multifaceted dimensions of the current diphtheria outbreak, which started in May 2022, juxtaposed against historical outbreaks, with a focal examination of the pervasive vaccine hesitancy and its underpinning sociocultural and systemic determinants. The discourse extends to a meticulous evaluation of Nigeria's public health response, underlined by the synergy with international organizations, reflecting a global collaborative ethos in combating the diphtheria menace. A critical appraisal of the prevailing immunization policies unveils a necessity for strategic amendments to invigorate vaccination uptake, essential for curbing the diphtheria outbreak and enhancing public health resilience. The reflections herein advocate for a comprehensive, culturally resonant, and sustainable public health paradigm, encompassing a synergistic approach of policy fortification, community engagement, and international collaboration to navigate the challenges posed by vaccine-preventable diseases epitomized by the ongoing diphtheria outbreak. Through a synthesis of historical lessons, contemporary challenges, and global solidarity, this piece contributes to the broader discourse on enhancing immunization coverage and infectious disease control in Nigeria.

Fighting diphtheria in Nigeria: the challenge of getting everyone vaccinated Recently, Nigeria has faced its worst diphtheria outbreak in years, showing how important it is to get people vaccinated. This article looks closely at the current outbreak, compares it to past ones, and explores why some people are hesitant to get vaccinated. We discuss the reasons behind this hesitation, including cultural and social factors. We also review how Nigeria is responding to the outbreak with help from international groups. We point out that Nigeria's vaccination policies need to be updated to encourage more people to get vaccinated, which is crucial to stop the outbreak and strengthen overall health defenses. The article calls for a well-rounded and lasting approach to public health that includes better policies, working closely with communities, and international cooperation.

Dengue virus type 1 clade replacement in recurring homotypic outbreaks.

BACKGROUND: Recurring dengue outbreaks occur in cyclical pattern in most endemic countries. The recurrences of dengue virus (DENV) infection predispose the population to increased risk of contracting the severe forms of dengue. Understanding the DENV evolutionary mechanism underlying the recurring dengue outbreaks has important implications for epidemic prediction and disease control. RESULTS: We used a set of viral envelope (E) gene to reconstruct the phylogeny of DENV-1 isolated between the periods of 1987-2011 in Malaysia. Phylogenetic analysis of DENV-1 E gene revealed that genotype I virus clade replacements were associated with the cyclical pattern of major DENV-1 outbreaks in Malaysia. A total of 9 non-conservative amino acid substitutions in the DENV-1 E gene consensus were identified; 4 in domain I, 3 in domain II and 2 in domain III. Selection pressure analyses did not reveal any positively selected codon site within the full length E gene sequences (1485 nt, 495 codons). A total of 183 (mean dN/dS = 0.0413) negatively selected sites were found within the Malaysian isolates; neither positive nor negative selection was noted for the remaining 312 codons. All the viruses were cross-neutralized by the respective patient sera suggesting no strong support for immunological advantage of any of the amino acid substitutions. CONCLUSION: DENV-1 clade replacement is associated with recurrences of major DENV-1 outbreaks in Malaysia. Our findings are consistent with those of other studies that the DENV-1 clade replacement is a stochastic event independent of positive selection.

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification.

BACKGROUND: Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic. METHODS: A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). RESULTS: Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses. CONCLUSION: The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

Extract of Scutellaria baicalensis inhibits dengue virus replication.

BACKGROUND: Scutellaria baicalensis (S. baicalensis) is one of the traditional Chinese medicinal herbs that have been shown to possess many health benefits. In the present study, we evaluated the in vitro antiviral activity of aqueous extract of the roots of S. baicalensis against all the four dengue virus (DENV) serotypes. METHODS: Aqueous extract of S. baicalensis was prepared by microwave energy steam evaporation method (MEGHE™), and the anti-dengue virus replication activity was evaluated using the foci forming unit reduction assay (FFURA) in Vero cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the actual dengue virus RNA copy number. The presence of baicalein, a flavonoid known to inhibit dengue virus replication was determined by mass spectrometry. RESULTS: The IC(50) values for the S. baicalensis extract on Vero cells following DENV adsorption ranged from 86.59 to 95.19 µg/mL for the different DENV serotypes. The IC(50) values decreased to 56.02 to 77.41 µg/mL when cells were treated with the extract at the time of virus adsorption for the different DENV serotypes. The extract showed potent direct virucidal activity against extracellular infectious virus particles with IC(50) that ranged from 74.33 to 95.83 µg/mL for all DENV serotypes. Weak prophylactic effects with IC(50) values that ranged from 269.9 to 369.8 µg/mL were noticed when the cells were pre-treated 2 hours prior to virus inoculation. The concentration of baicalein in the S. baicalensis extract was ~1% (1.03 µg/gm dried extract). CONCLUSIONS: Our study demonstrates the in vitro anti-dengue virus replication property of S. baicalensis against all the four DENV serotypes investigated. The extract reduced DENV infectivity and replication in Vero cells. The extract was rich in baicalein, and could be considered for potential development of anti-DENV therapeutics.

Tuberculosis treatment outcomes among pulmonary TB patients attending public hospitals in Kebbi State, Northern Nigeria: a four-year retrospective study.

Background: In Nigeria, effective case management and evaluation of pulmonary tuberculosis treatment outcomes are an integral part of controlling the spread of infectious diseases. The study reviewed the treatment outcomes of pulmonary tuberculosis and the factors associated with rates of successful and unsuccessful treatment outcomes in the 21 referral hospitals in Kebbi State, Nigeria. Methods: Documented records of pulmonary tuberculosis patients from January 2018 to December 2021 in 21 Local Area Councils in Kebbi State, Northern Nigeria were reviewed. A structured questionnaire collated the socio-demographic and clinical data from the documented records. Descriptive statistics were used to compute and analyse the outcomes of successful and unsuccessful treatment. Logistic regression models were used to determine the association of socio-demographic and clinical data with the unsuccessful treatment outcomes. Results: The study reviewed data from 6114 records of TB patients. 1161 (18.9%) started treatment, 963 (82.9%) were males and 198 (17.1%) were females. Of the 1161 patients, 985 (18.2%) had documented treatment outcomes. 932 of 985 (95.1%) had a pulmonary infection. 64 (5.8%) patients with documented treatment outcomes were HIV seropositive. 903 (91.7%) were successfully treated, and 82 (8.3%) failed. Of the patients with failed treatment outcomes, 15 (1.5%) were lost to follow-up, 43 (4.4%) defaulted and 24 (2.4%) died. In the logistic analysis, the odds of unsuccessful treatment outcomes were higher among elderly patients (AOR = 2.00, 95% CI 1.37-2.92), patients with extrapulmonary infections (AOR = 2.40, 95% CI 1.12-5.39), and with old cases of pulmonary TB (AOR = 3.03, 95% CI 1.47-7.19) when compared to their groups. Conclusions: The study reported a treatment success rate of 91.7% among TB patients attending public hospitals in Kebbi State. The outcome was higher than the projected success rate of 85% set by the WHO. However, one-fourth of the total patients reviewed were not documented for treatment. Therefore, the need to design an appropriate recruitment strategy to identify and enrol those patients for an effective and successful TB control program in Nigeria.

INCIDENCE OF RIFAMPICIN-RESISTANCE PRESUMPTIVE M. TUBERCULOSIS CASES AMONG OUTPATIENTS IN KEBBI STATE, NIGERIA.

BACKGROUND: The present study determined the incidence of rifampicin resistance M. tuberculosis among outpatients at the General Hospital Yauri, Kebbi State, Nigeria. MATERIALS AND METHODS: The study is a cross-sectional study conducted from February 2018 to October 2019. Sociodemographic data were collected from hospital registration books. Rifampicin resistance M. tuberculosis was detected using GeneXpert Model GX-IV following manufacturers' instruction. Descriptive statistics and logistic regression were computed using SPSS version 20. The results were presented as odds ratios with associated 95% confidence intervals, and P-value at 0.05. RESULT: Of the 837 samples, 65.8% (551/837) were males, and 34.2% (286/837) females, 11.4% (95/837) HIV-seropositive. M. tuberculosis was detected in 15.5% (130/837), of which 116/130 (89.23%) were males and 14/130 (10.77%) females. M. tuberculosis-HIV coinfection was detected in 9.47% (9/95) of HIV positive. Rifampicin resistance was observed in 1.3% (11/837), 7.7% (10/130) in M. tuberculosis patients and 1.05% (1/94) in HIV seropositive. In logistic regression, the odds ratio for having a rifampicin-resistant M. tuberculosis was 0.49 (0.15-1.54) for > 30 years; taking <30 years as the reference value, 1.02 (1.00-1.03) for male; taking female as the reference value, and 0.78 (0.09-6.15) for HIV positive, taking negative as the reference value. CONCLUSION: This study reported the current incidence rate of rifampicin-resistant M. tuberculosis at the General Hospital Yelwa Yauri, Kebbi State, Nigeria, among presumptive TB patients. Patients diagnosed with rifampicin-resistant M. tuberculosis were predominantly male adults. Thus, frequent screening is vital for surveillance and reduces the risk of transmission and spread of M. tuberculosis infections.