Testing In Resource-limited Settings Research Articles

Performance evaluation of loop-mediated isothermal amplification, polymerase chain reaction and real-time polymerase chain reaction methods to detect Neisseria gonorrhoeae among symptomatic patients from India.

Background: Neisseria gonorrhoeae is one of the most important causative organisms in causing sexually transmitted infections. The clinical presentation of gonorrhoea mimics the symptoms of other sexually transmitted infections, and a proper diagnosis of the same is therefore crucial in patient management. The current study intended to compare different in-house molecular methods: that is, conventional PCR, real-time PCR, and LAMP assay for detection of N. gonorrhoeae. Methods: A total of 163 samples were collected from 145 patients who presented with urethral and vaginal discharge. Collected samples were processed for culture on GC agar base, and three different molecular diagnostic tests (conventional PCR, real-time PCR, and LAMP assay) were performed simultaneously on all the samples. Results: Culture of N. gonorrhoeae was positive in 17 out of 21 (80.9%) swab samples. With culture as the gold standard method, conventional and real-time PCR had a sensitivity of 94.1%, whereas the sensitivity of the LAMP assay was found to be 88.2%. All three methods had a specificity of 100%. In addition to swab samples, evaluation of urine samples by different molecular methods yielded a good concordance with a kappa value of 0.85 by conventional PCR and real-time PCR showing a perfect level of agreement, while the LAMP assay was found to have a substantial level of agreement. Conclusion: LAMP assay had a comparable diagnostic accuracy to other molecular methods for the detection of N. gonorrhoeae and can be used as a point-of-care test in resource-limited settings.

Performance of two SARS-CoV-2 rapid antigen detection tests in resource limited settings, the case of Mali

Introduction: While real-time reverse transcription PCR (RT-PCR) is the recommended laboratory method to diagnose severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection, its use in resource limited settings can be difficult to maintain due to high testing demand and shortage of reagents. The aim of this study was to evaluate the performances of Realy Tech™ and Standard Q™ in comparison to RT-PCR in a relatively low COVID-19 prevalence setting, Mali.Methods: We conducted a cross-sectional study between January and April 2021 in Bamako and Kati regions to evaluate both rapid tests during a large SARS-CoV-2 prevalence study in Mali.Results: Of the 390 samples tested, the sensitivity and specificity of Realy Tech™ and Standard Q™ were 57.1% (95%CI: 44.1-69.2), 95.8% (95%CI: 93.1-97.5); 61.9% (95%CI: 46.8-75.0), and 94.1% (95%CI: 89.5-96.8) respectively. Using RT-PCR, the global prevalence of SARS-CoV-2 was 14.4% (56/390). In both rapid antigen tests, the performance was better when used in suspected patients compared to positive patients under treatment. Moreover, higher viral loads equivalent to Ct < 25 were associated with better detection rates.Conclusion: While waiting for more complete data, these preliminary studies suggest that Realy Tech™ and Standard Q™ should not be used alone for COVID-19 diagnosis in Mali.

Boosting Electrochemiluminescence Immunoassay Sensitivity via Co-Pt Nanoparticles within a Ti3C2 MXene-Modified Single Electrode Electrochemical System on Raspberry Pi.

Point-of-care testing plays a crucial role in diagnostics within resource-poor areas, necessitating the utilization of portable and user-friendly devices. The adaptation of biosensors for point-of-care applications requires careful considerations, such as miniaturization, cost-effectiveness, and streamlined sample processing. In recent years, the electrochemiluminescence (ECL) immunoassay has gained significant attention due to its visual detection capabilities and ability to facilitate high-throughput analysis. However, the development of a practical and cost-effective ECL device remains a challenging task. This study presents the development of an integrated MXene-modified single-electrode electrochemical system (SEES) for visual and high-throughput ECL immunoassays incorporating a Raspberry Pi system. The SEES was designed by affixing a plastic sticker with multiple perforations onto a single carbon ink screen-printed electrode, which operates based on a resistance-induced potential difference. Leveraging the excellent adsorption and bioaffinity properties of the carbon ink screen-printed electrode, effective immobilization of antibodies was achieved. Furthermore, the incorporation of Co-Pt nanoparticles enhanced the ECL intensity and electron transfer kinetics, enabling the sensitive detection of SARS-CoV-2. The developed system comprised 18 individual reaction cells, allowing for simultaneous analysis while maintaining sample isolation. Impressively, the system achieved a remarkable minimum virus detection limit of 10-14 g mL-1, accompanied by a high R2 value of 0.9798. These findings highlight the promising potential of our developed system for efficient point-of-care testing in resource-limited settings.

Impact of storage time in dried blood samples (DBS) and dried plasma samples (DPS) for point-of-care hepatitis C virus (HCV) RNA quantification and HCV core antigen detection.

The scale-up of hepatitis C virus (HCV) diagnosis and treatment requires affordable and simple tools to improve access to care, especially in low- and middle-income settings with limited infrastructure or high-risk populations. Dried blood and plasma samples (DBS and DPS) are useful alternative for hepatitis C detection in settings lacking adequate infrastructure. We evaluated the performance of DBS and DPS vs plasma in a point-of-care HCV RNA quantitative assay (Xpert HCV Viral Load-Cepheid), and compared HCV core antigen (HCVcAg) detection by the Architect HCV core antigen assay (Abbott) in DBS vs serum. The dried samples were stored at room temperature for different storage times to reproduce the time from sampling to testing in settings with centralized diagnosis or when testing mobile populations. HCV RNA quantification in DBS and DPS presented 100% sensitivity and specificity and a high correlation for up to 3 months of storage. HCV viremia showed a mean decrease of 0.5 log10 IU/mL (DBS) and 0.3 log10 IU/mL (DPS) for storage times up to 1 month. Architect HCVcAg detection presented high sensitivity/specificity (96%/100%) in DBS tested immediately after sampling, decreasing to 86% sensitivity after 7 days of storage. However, sensitivity increased when an optimized cut-off was applied for each storage time. We conclude that DBS and DPS are suitable samples for HCV RNA detection and quantification, being DPS more reliable for shorter storage times. DBS can be also used for HCVcAg qualitative detection and the sensitivity can be increased when adjusting the cut-off values. IMPORTANCE Hepatitis C infection remains a global burden despite the effectiveness of antivirals. In the WHO roadmap to accomplish HCV elimination by 2030, HCV diagnosis is one of the main targets. However, identifying patients in resource-limited settings and high-risk populations with limited access to healthcare remains a challenge and requires innovative approaches that allow decentralized testing. The significance of our research is in verifying the good performance of dried samples for HCV diagnosis using two different diagnostics assays and considering the effect of room temperature storage in this sample format. We confirmed dried samples are an interesting alternative for HCV screening and reflex testing in resource-limited settings or high-risk populations.

Detection of acute dengue virus infection, with and without concurrent malaria infection, in a cohort of febrile children in Kenya, 2014-2019, by clinicians or machine learning algorithms.

Poor access to diagnostic testing in resource limited settings restricts surveillance for emerging infections, such as dengue virus (DENV), to clinician suspicion, based on history and exam observations alone. We investigated the ability of machine learning to detect DENV based solely on data available at the clinic visit. We extracted symptom and physical exam data from 6,208 pediatric febrile illness visits to Kenyan public health clinics from 2014-2019 and created a dataset with 113 clinical features. Malaria testing was available at the clinic site. DENV testing was performed afterwards. We randomly sampled 70% of the dataset to develop DENV and malaria prediction models using boosted logistic regression, decision trees and random forests, support vector machines, naïve Bayes, and neural networks with 10-fold cross validation, tuned to maximize accuracy. 30% of the dataset was reserved to validate the models. 485 subjects (7.8%) had DENV, and 3,145 subjects (50.7%) had malaria. 220 (3.5%) subjects had co-infection with both DENV and malaria. In the validation dataset, clinician accuracy for diagnosis of malaria was high (82% accuracy, 85% sensitivity, 80% specificity). Accuracy of the models for predicting malaria diagnosis ranged from 53-69% (35-94% sensitivity, 11-80% specificity). In contrast, clinicians detected only 21 of 145 cases of DENV (80% accuracy, 14% sensitivity, 85% specificity). Of the six models, only logistic regression identified any DENV case (8 cases, 91% accuracy, 5.5% sensitivity, 98% specificity). Without diagnostic testing, interpretation of clinical findings by humans or machines cannot detect DENV at 8% prevalence. Access to point-of-care diagnostic tests must be prioritized to address global inequities in emerging infections surveillance.

The risk of sexual transmission of HIV in individuals with low-level HIV viraemia: a systematic review

The risk of sexual transmission of HIV from individuals with low-level HIV viraemia receiving antiretroviral therapy (ART) has important public health implications, especially in resource-limited settings that use alternatives to plasma-based viral load testing. This Article summarises the evidence related to sexual transmission of HIV at varying HIV viral load levels to inform messaging for people living with HIV, their partners, their health-care providers, and the wider public. We conducted a systematic review and searched PubMed, MEDLINE, Cochrane Central Register of Controlled Trials, Embase, Conference Proceedings Citation Index-Science, and WHO Global Index Medicus, for work published from Jan 1, 2010 to Nov 17, 2022. Studies were included if they pertained to sexual transmission between serodiscordant couples at various levels of viraemia, the science behind undetectable=untransmittable, or the public health impact of low-level viraemia. Studies were excluded if they did not specify viral load thresholds or a definition for low-level viraemia or did not provide quantitative viral load information for transmission outcomes. Reviews, non-research letters, commentaries, and editorials were excluded. Risk of bias was evaluated using the ROBINS-I framework. Data were extracted and summarised with a focus on HIV sexual transmission at varying HIV viral loads. 244 studies were identified and eight were included in the analysis, comprising 7762 serodiscordant couples across 25 countries. The certainty of evidence was moderate; the risk of bias was low. Three studies showed no HIV transmission when the partner living with HIV had a viral load less than 200 copies per mL. Across the remaining four prospective studies, there were 323 transmission events; none were in patients considered stably suppressed on ART. Among all studies there were two cases of transmission when the index patient's (ie, patient with previously diagnosed HIV infection) most recent viral load was less than 1000 copies per mL. However, interpretation of both cases was complicated by long intervals (ie, 50 days and 53 days) between the transmission date and the most recent index viral load result. There is almost zero risk of sexual transmission of HIV with viral loads of less than 1000 copies per mL. These data provide a powerful opportunity to destigmatise HIV and promote adherence to ART through dissemination of this positive public health message. These findings can also promote access to viral load testing in resource-limited settings for all people living with HIV by facilitating uptake of alternative sample types and technologies. Bill & Melinda Gates Foundation.

Descriptive spectrum of thiamine deficiency in pregnancy: Apotentially preventable condition.

Pregnancy, a nutritionally demanding situation in terms of macro- and micronutrient supply owing to heightened maternal, placental, and fetal needs, significantly affects thiamine reserves. Thiamine deficiency during pregnancy and the postpartum period, presenting with varied manifestations and outcomes, is a relatively common condition in our population. The study aimed to understand the various manifestations and outcomes of acute thiamine deficiency in pregnant and postpartum women, emphasizing the significance of early recognition and thiamine therapy to prevent serious complications during pregnancy and after childbirth. This prospective study conducted in a tertiary care center in North India enrolled consecutive pregnant and postpartum women presenting with clinical features consistent with thiamine deficiency disorders, such as thiamine deficiency-related neuropathy, high-output heart failure, heart failure with reduced ejection fraction, Wernicke's encephalopathy, gastric beriberi, and thiamine-responsive acute pulmonary hypertension. In addition to capturing medical history including drug intake, dietary consumption, and comorbidities, women underwent brief relevant clinical examinations and laboratory assessments, including whole-blood thiamine levels. Response to intravenous thiamine supplementation was also monitored. Data of 31 women (12 pregnant, 19 postpartum) with a diagnosis of acute thiamine deficiency and a mean age of 28.88 ± 2.69 years were analyzed. The mean thiamine level was 1.28 ± 0.44 μg/dL with mean blood lactate of 3.46 ± 3.33. The most common presentation was gastric beriberi (n = 10), followed by paraparesis (n = 6), high-output heart failure (n = 6), acute pulmonary hypertension, heart failure with reduced ejection fraction (n = 3 each), and an acute confusional state (n = 2). All patients responded to thiamine challenge. In the context of borderline thiamine status, particularly in our population with endemic thiamine deficiency and heightened demand for thiamine during pregnancy and the peripartum period, the deficiency can have varied and serious manifestations of dry and wet beriberi. Early recognition of the clinical features and thiamine therapy can be life-saving. There is a need for validated clinical criteria owing to the non-availability of thiamine testing in resource-limited settings.

Comparison of methods to engage diverse stakeholder populations in prioritizing PrEP implementation strategies for testing in resource-limited settings: a cross-sectional study

BackgroundThere is a lack of consensus about how to prioritize potential implementation strategies for HIV pre-exposure prophylaxis (PrEP) delivery. We compared several prioritization methods for their agreement and pragmatism in practice in a resource-limited setting.MethodsWe engaged diverse stakeholders with clinical PrEP delivery and PrEP decision-making experience across 55 facilities in Kenya to prioritize 16 PrEP delivery strategies. We compared four strategy prioritization methods: (1) “past experience surveys” with experienced practitioners reflecting on implementation experience (N = 182); (2 and 3) “pre- and post-small-group ranking” surveys before and after group discussion (N = 44 and 40); (4) “go-zone” quadrant plots of perceived effectiveness vs feasibility. Kendall’s correlation analysis was used to compare strategy prioritization using the four methods. Additionally, participants were requested to group strategies into three bundles with up to four strategies/bundle by phone and online survey.ResultsThe strategy ranking correlation was strongest between the pre- and post-small-group rankings (Tau: 0.648; p < 0.001). There was moderate correlation between go-zone plots and post-small-group rankings (Tau: 0.363; p = 0.079) and between past-experience surveys and post-small-group rankings (Tau: 0.385; p = 0.062). For strategy bundling, participants primarily chose bundles of strategies in the order in which they were listed, reflecting option ordering bias. Neither the phone nor online approach was effective in selecting strategy bundles. Participants agreed that the strategy ranking activities conducted during the workshop were useful in prioritizing a final set of strategies.ConclusionsBoth experienced and inexperienced stakeholder participants’ strategy rankings tended to prioritize strategies perceived as feasible. Small group discussions focused on feasibility and effectiveness revealed moderately different priorities than individual rankings. The strategy bundling approach, though less time- and resource-intensive, was not effective. Future research should further compare the relative effectiveness and pragmatism of methodologies to prioritize implementation strategies.

Near point-of-care HIV viral load testing: Cascade after high viral load in suburban Yangon, Myanmar.

HIV viral load (VL) testing in resource-limited settings is often centralised, limiting access. In Myanmar, we assessed outcomes according to VL access and the VL cascade (case management after a first high VL result) before and after near point-of-care (POC) VL was introduced. Routine programme data from people living with HIV (PLHIV) on antiretroviral therapy (ART) were used. We assessed the odds of getting a VL test done by year. Attrition and mortality two years after ART initiation were compared between three groups of PLHIV with different access to VL testing using Kaplan-Meier analysis. We compared VL cascades in those with a first VL result before and after near POC VL testing became available. With logistic regression, predictors of confirmed virological failure after a first high VL in the POC era were explored. Among 4291 PLHIV who started ART between July 2009 and June 2018, 794 (18.5%) became eligible for VL testing when it was not available, 2388 (55.7%) when centralised laboratory-based VL testing was available, and 1109 (25.8%) when near POC VL testing was available. Between 2010 and 2019, the odds of getting a VL test among those eligible increased with each year (OR: 5.21 [95% CI: 4.95-5.48]). Attrition and mortality were not different in the three groups. When comparing PLHIV with a first VL result before and after implementation of the near POC VL testing, in the latter, more had a first VL test (92% versus 15%, p<0.001), less had a first high VL result (5% versus 14%, p<0.001), and more had confirmed virological failure (67% versus 47%, p = 0.013). Having a first VL ≥5000 copies/mL after near POC implementation was associated with confirmed virological failure (adjusted OR: 2.61 [95% CI: 1.02-6.65]). Near POC VL testing enabled rapid increase of VL coverage and a well-managed VL cascade in Myanmar.

The use of colorimetric loop-mediated isothermal amplification assay for naked-eye detection of bean common mosaic virus

Bean common mosaic virus (BCMV), one of the most prevalent viral diseases of common beans, has been intensively studied since its initial identification. In this paper, a one-step colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method was performed by designing oligonucleotide primers to amplify the gene encoding the coat protein of BCMV. Positive LAMP reactions were observed with the naked eye using WarmStart 2 × Master Mix from NEB, changing from pink to yellowish if the test is positive. The sensitivity of this method was similar to that of conventional PCR. The method was tested on common bean and another virus, bean common mosaic necrosis virus (BCMNV), and it was shown to specifically detect BCMV and not produce false positive from other RNAs. The RT-LAMP test was quite reliable for the diagnosis of BCMV from infected samples, and the precise, sensitive. The cost analysis showed that the development of a less expensive and efficient RT-LAMP assay for BCMV detection is an important step in expanding testing capacity and improving access to accurate testing in resource-limited settings. The test can be utilized in laboratory and in the field for the diagnosis and monitoring of BCMV, and is the first reported use of RT-LAMP for BCMV detection.

Utility of a Novel Clinical Scoring System and its Association with Procalcitonin in Patients of Acute Heart Failure with Respiratory Infections: A Limited Resource Perspective

Abstract Background: Heart failure (HF) is a global health burden that is usually simple to diagnose, but concomitant respiratory infections are difficult to distinguish clinically. Inflammatory markers can be used to differentiate between the two conditions, especially procalcitonin (PCT), one of the most specific markers for bacterial infections. PCT-guided antibiotic treatment has shown good results and could help in decreasing the indiscriminate prescription of antibiotics in HF patients. Methodology: A prospective observational study was carried out in a tertiary care hospital in Western India. Three hundred HF patients were separated into either high- or low-PCT level groups. A novel clinical score for clinically suspecting superimposed respiratory infection in acute HF patients was devised and its association with PCT level was measured. Results: 62.87 years was the average age of the study population with a male preponderance. While breathlessness was the presenting complaint in all 300 patients, the presence of fever and edema was significantly associated with raised PCT. A higher total leukocyte count and neutrophil count were seen in patients with high PCT. A higher incidence of consolidation, pulmonary edema, and pulmonary hypertension was noted in those with high PCT. A significant association was found between the clinical scoring system and the PCT level. Conclusion: The novel clinical scoring system has a satisfactory sensitivity, specificity, positive predictive value, and negative predictive value in diagnosing superimposed respiratory infections in acute HF patients. It can aid in the judicious use of the PCT test in resource-limited settings.

Improving awareness, diagnosis and management of invasive fungal infections in Ghana: establishment of the Ghana Medical Mycology Society.

Invasive fungal infections (IFIs) and medical mycology receive little attention in Ghana. However, the present evolution of biomarker assays for IFIs, offers an opportunity for an increased access to fungal laboratory testing in resource-limited settings, and probably make a case for availability of essential antifungal agents. Using surveys and personal communications, the state of medical mycology and IFI in Ghana were highlighted. Inadequate awareness and insufficient access to fungal diagnostics and therapeutics were identified as the key challenges, the establishment of the Ghana Medical Mycology Society was discussed, and recommendations were made to improve the status quo.

The performance of using dried blood spot specimens for HIV-1 viral load testing: A systematic review and meta-analysis.

BackgroundAccurate routine HIV viral load testing is essential for assessing the efficacy of antiretroviral treatment (ART) regimens and the emergence of drug resistance. While the use of plasma specimens is the standard for viral load testing, its use is restricted by the limited ambient temperature stability of viral load biomarkers in whole blood and plasma during storage and transportation and the limited cold chain available between many health care facilities in resource-limited settings. Alternative specimen types and technologies, such as dried blood spots, may address these issues and increase access to viral load testing; however, their technical performance is unclear. To address this, we conducted a meta-analysis comparing viral load results from paired dried blood spot and plasma specimens analyzed with commonly used viral load testing technologies.Methods and findingsStandard databases, conferences, and gray literature were searched in 2013 and 2018. Nearly all studies identified (60) were conducted between 2007 and 2018. Data from 40 of the 60 studies were included in the meta-analysis, which accounted for a total of 10,871 paired dried blood spot:plasma data points. We used random effects models to determine the bias, accuracy, precision, and misclassification for each viral load technology and to account for between-study variation. Dried blood spot specimens produced consistently higher mean viral loads across all technologies when compared to plasma specimens. However, when used to identify treatment failure, each technology compared best to plasma at a threshold of 1,000 copies/ml, the present World Health Organization recommended treatment failure threshold. Some heterogeneity existed between technologies; however, 5 technologies had a sensitivity greater than 95%. Furthermore, 5 technologies had a specificity greater than 85% yet 2 technologies had a specificity less than 60% using a treatment failure threshold of 1,000 copies/ml. The study’s main limitation was the direct applicability of findings as nearly all studies to date used dried blood spot samples prepared in laboratories using precision pipetting that resulted in consistent input volumes.ConclusionsThis analysis provides evidence to support the implementation and scale-up of dried blood spot specimens for viral load testing using the same 1,000 copies/ml treatment failure threshold as used with plasma specimens. This may support improved access to viral load testing in resource-limited settings lacking the required infrastructure and cold chain storage for testing with plasma specimens.

Recent Advances in Molecular Point of Care Tests

Molecular point of care tests has become promising methods for the global control of infectious diseases in recent years. Different kinds of point of care testing devices have been introduced into the market in the last decade. They are mainly based on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. These devices must offer a low-cost, sensitive, specific, and practical analysis to be used especially in low-resource settings. Microfluidics has high potential for handling very small volumes of fluids on a single platform. The key design features for molecular point of care tests in resource-limited settings include isothermal nucleic acid amplification methods to eliminate the need for a thermocycler, lyophilized reagents for long-term stability at high temperature and relatively simple test procedures. CRISPR-Cas-based new generation molecular point of care tests have been developed in recent years. In these tests Cas enzymes are used as highly specific target sequence recognition elements. Collateral cleavage activity of these enzymes cleaves both target sequence and labeled RNA in the mixture and a signal is generated. The ongoing Coronavirus 2019 pandemic has shown the importance of rapid diagnostic tests for the prevention of further spread. Although real-time polymerase chain reaction method was used successfully for the rapid diagnosis during the pandemic, additional rapid and practical tests that could be performed outside the laboratories would provide even faster diagnosis and lighten the burden of test load in large central laboratories. Molecular point of care tests are considered to be one of the best candidates to fill the this gap in the near future. The future technology challenges will be the standardization of the methods and further miniaturization of the system components. In this review article, point-of-care tests adapted from nucleic acid amplification methods with a large number of studies in the literature and tests based on the CRISPR-Cas system which constitutes the newest group and which is among the point-of-care molecular tests based on new nextgeneration sequencing have been discussed.

Direct Processing and Storage of Cell-Free Plasma Using Dried Plasma Spot Cards.

Plasma separation cards represent a viable approach for expanding testing capabilities away from clinical settings by generating cell-free plasma with minimal user intervention. These devices typically comprise a basic structure of the plasma separation membrane, unconstrained porous collection pad, and utilize either (i) lateral or (ii) vertical fluidic pathways for separating plasma. Unfortunately, these configurations are highly susceptible to (i) inconsistent sampling volume due to differences in the patient hematocrit or (ii) severe contamination due to leakage of red blood cells or release of hemoglobin (i.e., hemolysis). Herein, we combine the enhanced sampling of our previously reported patterned dried blood spot cards with an assembly of porous separation materials to produce a patterned dried plasma spot card for direct processing and storage of cell-free plasma. Linking both vertical separation and lateral distribution of plasma yields discrete plasma collection zones that are spatially protected from potential contamination due to hemolysis and an inlet zone enriched with blood cells for additional testing. We evaluate the versatility of this card by quantitation of three classes of analytes and techniques including (i) the soluble transferrin receptor by enzyme-linked immunosorbent assay, (ii) potassium by inductively coupled plasma atomic emission spectroscopy, and (iii) 18S rRNA by reverse transcriptase quantitative polymerase chain reaction. We achieve quantitative recovery of each class of analyte with no statistically significant difference between dried and liquid reference samples. We anticipate that this sampling approach can be applied broadly to improve access to critical blood testing in resource-limited settings or at the point-of-care.

Cervical cancer screening programmes and age-specific coverage estimates for 202 countries and territories worldwide: a review and synthetic analysis

SummaryBackgroundCervical cancer screening coverage is a key monitoring indicator of the WHO cervical cancer elimination plan. We present global, regional, and national cervical screening coverage estimates against the backdrop of the 70% coverage target set by WHO.MethodsIn this review and synthetic analysis, we searched scientific literature, government websites, and official documentation to identify official national recommendations and coverage data for cervical cancer screening for the 194 WHO member states and eight associated countries and territories published from database inception until Oct 30, 2020, supplemented with a formal WHO country consultation from Nov 27, 2020, to Feb 12, 2021. We extracted data on the year of introduction of recommendations, the existence of individual invitation to participate, financing of screening tests, primary screening and triage tests used, recommended ages and screening intervals, use of self-sampling, and use of screen-and-treat approaches. We also collected coverage data, either administrative or survey-based, as disaggregated as possible by age and for any available screening interval. According to data completeness and representativeness, different statistical models were developed to produce national age-specific coverages by screening interval, which were transformed into single-age datapoints. Missing data were imputed. Estimates were applied to the 2019 population and aggregated by region and income level.FindingsWe identified recommendations for cervical screening in 139 (69%) of 202 countries and territories. Cytology was the primary screening test in 109 (78%) of 139 countries. 48 (35%) of 139 countries recommended primary HPV-based screening. Visual inspection with acetic acid was the most recommended test in resource-limited settings. Estimated worldwide coverage in women aged 30–49 years in 2019 was 15% in the previous year, 28% in the previous 3 years, and 32% in the previous 5 years, and 36% ever in lifetime. An estimated 1·6 billion (67%) of 2·3 billion women aged 20–70 years, including 662 million (64%) of 1·0 billion women aged 30–49 years, had never been screened for cervical cancer. 133 million (84%) of 158 million women aged 30–49 years living in high-income countries had been screened ever in lifetime, compared with 194 million (48%) of 404 million women in upper-middle-income countries, 34 million (9%) of 397 million women in lower-middle-income countries, and 8 million (11%) of 74 million in low-income countries.InterpretationTwo in three women aged 30–49 years have never been screened for cervical cancer. Roll-out of screening is very low in low-income and middle-income countries, where the burden of disease is highest. The priority of the WHO elimination campaign should be to increase both screening coverage and treatment of detected lesions; however, expanding the efforts of surveillance systems in both coverage and quality control are major challenges to achieving the WHO elimination target.FundingInstituto de Salud Carlos III, European Regional Development Fund, Secretariat for Universities and Research of the Department of Business and Knowledge of the Government of Catalonia, and Horizon 2020.TranslationsFor the French, Spanish translations of the abstract see Supplementary Materials section.

#64 Common Admission Diagnoses for Children Hospitalized with Respiratory Syncytial Virus and Antibiotic Administration Practices in Amman, Jordan

Abstract Background Determining the etiologic pathogen for acute respiratory infections is challenging, especially in low- and middle-income countries, where diagnostic testing is limited. These barriers may contribute to antibiotic overuse for viral infections. We aimed to evaluate the antibiotic administration patterns among hospitalized children with a research positive test for respiratory syncytial virus (RSV). Method Children admitted to a government-run hospital in Amman, Jordan with acute respiratory symptoms and/or fever from January 8 to March 17, 2020, were eligible for enrollment. Trained personnel conducted interviews and chart abstractions using a standardized data collection form. Nasal and/or throat swabs were collected and tested for RSV by PCR. Differences between the three most common admission diagnoses for RSV-positive children [bronchiolitis, bronchopneumonia (BP), and pneumonia] were evaluated. Results A total of 531 children were enrolled and tested and 401 (76%) were RSV-positive, of which 96% received an antibiotic and 25%, 26%, and 32% had an admission diagnosis of bronchiolitis, BP, or pneumonia, respectively. Compared to bronchiolitis and BP, RSV-positive children with a pneumonia were younger and more likely to require supplemental oxygen, receive an antibiotic during hospitalization, be admitted to the ICU; but were less likely to present with wheezing and to report antibiotic use prior to hospitalization (Table 1). RSV-positive children with BP were more likely to present with fever and have a higher C-reactive protein compared to the other diagnoses, whereas bronchiolitis was less likely to be administered antibiotics overall, but more likely to be given a macrolide. Conclusion Our results suggest that clinical presentation alone is insufficient in correctly determining causative pathogens that warrant empiric antibiotics. Thus, evaluating the role of point-of-care diagnostic testing in resource-limited settings is important and may help limit antibiotic use.

Performance characteristics of two new rapid HIV diagnostic assays and use of test band reader

PurposeAccurate HIV diagnosis is essential for appropriate patient care. This present study evaluated the performance of two different new rapid HIV diagnostic tests; 1) TRUSTline HIV-1/2 Ab rapid test (Athenese-DxPvt. Ltd, Chennai, India)and 2) OnSite HIV 1/2 Ab Plus Combo Rapid Test (CTK Biotech Inc., San Diego, USA) and also validated ALTA Rapid Test Reader (RTR-1) (CTK Biotech Inc., San Diego, USA), the device is a user-friendly and image-analysis based qualitative/semi-quantitative tabletop reader. MethodsA total of n ​= ​500 characterized specimens were used for this evaluation and the results of the new test kits (TRUSTline and OnSite) were also compared with 4th generation ELISA kit (Genescreen™Ultra HIV Ag-Ab ELISA) and 3 other commercially available rapid tests that were in the market; 1)SD Bioline™ HIV 1/2 3.0, 2) Aspen® HIV 1/2 Rapid Ab Test and 3) Diagnostic enterprises HIVTRI-DOT. The test band intensities of the TRUSTline and OnSite tests were measured in an ALTA rapid test reader and compared with the naked eye reading. ResultsThe sensitivity, specificity, positive predictive value, negative predictive value and efficacy of TRUSTline and OnSite were 100%, 99.6%, 99.5%, 100% and 99.8% and 100%, 100%, 100%, 100% and 100% respectively. ConclusionsThe ‘TRUSTline HIV-1/2’ and ‘OnSite HIV 1/2' kits are suitable to use in the HIV testing algorithm. Use of the ALTA rapid test reader could be user's friendly in the field level testing in resource-limited settings”.

Development and Testing of a Low-Cost Inactivation Buffer That Allows for Direct SARS-CoV-2 Detection in Saliva.

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

An optimization of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams.

Background:The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in a resource-limited setting. Methods:We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive - GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results:The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion:We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit's performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results.