Sample-to-result molecular diagnostic platforms and their suitability for infectious disease testing in low- and middle-income countries.

INTRODUCTION: Diagnostics are an essential, undervalued part of the health-care system. For many diseases, molecular diagnostics are the gold standard, but are not easy to implement in Low- and Middle-Income Countries (LMIC). Sample-to-result (S2R) platforms combining all procedures in a closed system could offer a solution. In this paper, we investigated their suitability for implementation in LMIC. AREAS COVERED: A scorecard was used to evaluate different platforms on a range of parameters. Most platforms scored fairly on the platform itself, ease-of-use and test consumables; however, shortcomings were identified in cost, distribution and test panels tailored to LMIC needs. The diagnostic coverage for common infectious diseases was found to have a wider coverage in high-income countries (HIC) than LMIC. A literature study showed that in LMIC, these platforms are mainly used as diagnostic tools or evaluation of diagnostic performance, with a minority assessing the operational characteristics or the clinical utility. In this narrative review, we identified various points for adaptation of S2R platforms to LMIC conditions. EXPERT OPINION: For S2R platforms to be suitable for implementation in LMIC some modifications by the manufacturers could be considered. Furthermore, strengthening health systems and digitalization are vital; as are smaller, cheaper, faster, and sustainable technologies.

Practical considerations for pathological diagnosis and molecular profiling of cholangiocarcinoma: an expert review for best practices.

INTRODUCTION: Advances in precision medicine have expanded access to targeted therapies and demand for molecular profiling of cholangiocarcinoma (CCA) patients in routine clinical practice. However, pathologists face challenges in establishing a definitive intrahepatic CCA (iCCA) diagnosis while preserving sufficient tissue for molecular profiling. Additionally, they frequently face challenges in optimal tissue handling to preserve nucleic acid integrity. AREAS COVERED: This article first identifies the challenges in establishing a definitive diagnosis of iCCA in a lesional liver biopsy while preserving sufficient tissue for molecular profiling. Then, the authors explore the clinical value of molecular profiling, the basic principles of single gene and next-generation sequencing (NGS) techniques, and the challenges in tissue sampling for genomic testing. They also propose an algorithm for best practice in tissue management for molecular profiling of CCA. EXPERT OPINION: Several practical challenges face pathologists during tissue sampling and processing for molecular profiling. Optimized tissue processing, careful tissue handling, and selection of appropriate approaches to molecular testing are essential to ensure that the highest possible quality of diagnostic information is provided in the greatest proportion of cases.

Development of advanced control material for reverse transcription-mediated bacterial nucleic acid amplification tests.

Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.

2023 global inventory of commercial molecular tests for human papillomaviruses (HPV).

To suit the needs of the human papillomaviruses (HPV) community comprehensively, a range of commercial HPV tests with different performance characteristics are required. Four periodic inventories of commercial HPV molecular tests present in the global market were published previously in 2010, 2012, 2015 and 2020. For the fifth inventory, data were retrieved from internal files and a detailed search using the main bibliographic databases as well as general internet search without period or language restrictions was performed in December 2023. At least 264 distinct HPV tests (and 511 test variants) were available globally in December 2023. A small 2020-2023 net increase in total numbers was observed, but with a strong introduction/withdrawal dynamic: 86 new distinct HPV tests (and 141 variants) were introduced and 76 tests (and 55 variants) were withdrawn from the market in the last four years. Although quality improvement of some tests was recorded, half of all HPV tests are still without a single peer-reviewed publication, and 79 % of tests are without published evidence that demonstrate performance characteristics are in line with requirements agreed in the HPV community. Only a relatively small pool of tests fulfill the operational/performance characteristics required to meet the global cervical cancer screening challenge. Although clinical and analytical performance characteristics of many commercial HPV tests are largely unknown, such tests are used worldwide in daily clinical practice and research, with potentially deleterious consequences. Due to this long-lasting unfavorable situation, significant scope for improvement persists for both manufacturers of HPV tests and the HPV community.

Validation of a rapid molecular detection test for gram-negative multidrug-resistant bacteria in rectal swabs upon admission of patients to the intensive care unit.

In ICU settings, screening patients upon admission for potential multiresistant bacteria (BMR) carriers is crucial. Traditionally, clinical decisions relied on delayed culture results, but a rapid PCR molecular test called RealCycler-Rezero-U/G (Progenie-molecular©), emerged as an alternative. This study aimed to validate its effectiveness in detecting gram-negative BMR in rectal swabs at ICU admission. Over 24 months, an observational study was conducted on 1,234 admitted patients, with 217 meeting isolation criteria and undergoing both PCR and culture tests. Results showed a 17.5 % positive rate for screening. The PCR test exhibited impressive accuracy at 98.6 % and a strong negative predictive value of 99.4 %. The area under the ROC curve (AUC) was 0.98, indicating high reliability. Notably, PCR results were available 44.5 h earlier than culture. In conclusion, PCR-based molecular testing for gram-negative BMR offers excellent diagnostic performance and a valuable negative predictive value, making it a suitable screening tool for ICU admissions.

Utility of quantitative loop mediated isothermal amplification (qLAMP) assay for the diagnosis of invasive pneumococcal disease (IPD).

PURPOSE: Quantitative LAMP (qLAMP) assay is one of the recent and emerging diagnostic tests for infectious diseases. Only a few studies exist comparing this assay with quantitative real-time PCR (qPCR) for the diagnosis of invasive pneumococcal disease (IPD). AIM: To compare the diagnostic performance of qLAMP assay with qPCR targeting autolysin gene for the diagnosis of invasive pneumococcal disease. METHODS: Ninety six blood samples and 73 CSF samples from patients clinically suspected with community acquired pneumonia and acute meningitis were tested by qPCR and qLAMP assays using previously published primers and protocols. The qPCR was considered as the gold standard test and the diagnostic performance was assessed by calculating sensitivity, specificity, positive and negative predictive values, and kappa coefficient for the level of agreement between the tests. Chi-squared/Fisher exact test was used to compare categorical variables (positive/negative). RESULTS: Thirty two blood samples and 22 CSF samples were positive by qPCR while 24 and 20 samples were positive by qLAMP assay respectively. The sensitivity of qLAMP assay was only 86.4% and 75% when tested on CSF and blood samples respectively. However, the qLAMP assay was in substantial to almost perfect agreement when compared with qPCR. The results were statistically significant in both sample types (P < 0.001). CONCLUSIONS: The performance of qLAMP assay can vary based on the specimen type. It has very high specificity and had substantial to almost perfect agreement, and thus may be an alternative to qPCR for the diagnosis of IPD.

New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Diagnostic utility of LAMP PCR targeting bcsp-31 gene for human brucellosis infection.

PURPOSE: Human brucellosis is a neglected zoonotic disease of significant public health concern. Molecular diagnosis of brucella remains challenging in low resource settings, due to the high infrastructure and cost involved. Loop-mediated isothermal amplification (LAMP) is a rapid point of care polymerase chain reaction (PCR) with the utility of on-field molecular diagnosis and offers a convenient alternative to conventional PCR. In the present study, we developed and evaluated the diagnostic utility of in house LAMP PCR targeting the Brucella genus-specific bcsp-31 gene in patients having febrile illness. MATERIALS AND METHODS: The analytical sensitivity and specificity of bcsp-31 LAMP PCR was first evaluated using brucella (n â€‹= â€‹8) and non-brucella cultures (n â€‹= â€‹5), along with spiked clinical samples. The overall diagnostic utility of developed LAMP PCR was then further evaluated in 393 human samples suspected of brucellosis. RESULTS: The developed LAMP PCR could detect as low as 8 â€‹fg of DNA by visual detection within 35min. We report sensitivity and specificity of the developed LAMP PCR as 90.91% and 99.37%.The accuracy of the developed test assay was found to be 98.60%. In clinical samples, LAMP gave positivity of 20% with the concordance of 89% with conventional PCR. CONCLUSION: To conclude, a rapid, efficacious, sensitive LAMP PCR targeting the bcsp 31 gene was developed. The existing LAMP PCR can be used as a point of care screening test in various low resource endemic setting in lieu of conventional PCR for estimation of prevalence data, diagnosis and treatment of brucellosis.

Molecular-Based Immunohistochemical Algorithm for Uterine Leiomyosarcoma Diagnosis.

The morphologic assessment of uterine leiomyosarcoma (LMS) may be challenging, and diagnostic immunohistochemical (IHC) analysis is currently lacking. We evaluated the genomic landscape of 167 uterine LMS by targeted next-generation sequencing (NGS) to identify common genomic alterations. IHC analyses corresponding to these genomic landmarks were applied to a test cohort of 16 uterine LMS, 6 smooth muscle tumors of uncertain malignant potential (STUMP), and 6 leiomyomas with NGS data and a validation cohort of 8 uterine LMS, 12 STUMP, 21 leiomyomas and leiomyoma variants, 7 low-grade endometrial stromal sarcomas, and 2 diagnostically challenging uterine smooth muscle tumors. IHC results were individually interpreted by 3 pathologists blinded to NGS data. Overall, 94% of LMS showed ≥1 genomic alteration involving TP53, RB1, ATRX, PTEN, CDKN2A, or MDM2, with 80% showing alterations in ≥2 of these genes. In the test cohort, an initial panel of p53, Rb, PTEN, and ATRX was applied, followed by a panel of DAXX, MTAP, and MDM2 in cases without abnormalities. Abnormal p53, Rb, PTEN, and ATRX IHC expression was seen in 75%, 88%, 44%, and 38% of LMS, respectively, in the test cohort. Two or more abnormal IHC results among these markers were seen in 81% of LMS. STUMPs demonstrated only 1 IHC abnormality involving these markers. No IHC abnormalities were seen in leiomyomas. In the validation cohort, abnormal p53, Rb, and PTEN IHC results were seen in LMS, whereas rare STUMP or leiomyomas with bizarre nuclei showed IHC abnormalities involving only 1 of the markers. Abnormalities in ≥2 markers were present in both diagnostically challenging smooth muscle tumors, confirming LMS. Concordance was excellent among pathologists in the interpretation of IHC (κ = 0.97) and between IHC and NGS results (κ = 0.941). Uterine LMS exhibit genomic landmark alterations for which IHC surrogates exist, and a diagnostic algorithm involving molecular-based IHC may aid in the evaluation of unusual uterine smooth muscle tumors.

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.

Development and Evaluation of a Molecular Hepatitis A Virus Assay for Serum and Stool Specimens.

Hepatitis A virus (HAV) is an emerging public health concern and there is an urgent need for ways to rapidly identify cases so that outbreaks can be managed effectively. Conventional testing for HAV relies on anti-HAV IgM seropositivity. However, studies estimate that 10-30% of patients may not be diagnosed by serology. Molecular assays that can directly detect viral nucleic acids have the potential to improve diagnosis, which is key to prevent the spread of infections. In this study, we developed a real-time PCR (RT-PCR) assay to detect HAV RNA for the identification of acute HAV infection. Primers were designed to target the conserved 5'-untranslated region (5'-UTR) of HAV, and the assay was optimized on both the Qiagen Rotor-Gene and the BD MAX. We successfully detected HAV from patient serum and stool samples with moderate differences in sensitivity and specificity depending on the platform used. Our results highlight the clinical utility of using a molecular assay to detect HAV from various specimen types that can be implemented in hospitals to assist with diagnostics, treatment and prevention.

Comparison of three molecular diagnostic assays for SARS-CoV-2 detection: Evaluation of analytical sensitivity and clinical performance.

BACKGROUND: Currently, SARS-CoV-2 RNA detection using real-time reverse-transcription PCR (rRT-PCR) is the standard diagnostic test for COVID-19 infection. Various rRT-PCR assays are currently used worldwide, targeting different genes of the SARS-CoV-2. Here, we compared the analytical sensitivity and clinical performance (sensitivity and specificity) of Allplex SARS-CoV-2/FluA/FluB/RSV assay (Seegene), Standard M nCoV real-time detection kit (SD Biosensor), and U-TOP COVID-19 detection kit (Seasun Biomaterials) for SARS-CoV-2 detection. METHODS: Two hundred and forty-nine nasopharyngeal swab samples were evaluated to compare the clinical performance of the rRT-PCR assays. For the analytical performance evaluation, two RNA controls with known viral loads-SARS-CoV-2 RNA control and SARS-COV-2 B.1.351 RNA control-were used to investigate the potential impact of SARS-CoV-2 variants, particularly the B.1.351 lineage. RESULTS: Limits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (Ct ) values of the two RNA controls were within 1.0 for each target in the rRT-PCR assays (0.05-0.73), without any prominent Ct value shift or dropouts in the SARS-COV-2 B.1.351 RNA control. Using the consensus criterion as the reference standard, 89 samples were positive, whereas 160 were negative. The overall clinical performance of rRT-PCR assays was comparable (sensitivity 98.88%-100%; specificity 99.38%-100%), whereas the sensitivities of each target gene were more variable. CONCLUSIONS: The three rRT-PCR assays showed comparable analytical sensitivity and clinical performance. The analytical and clinical sensitivities of each target gene were influenced more by the primer and probe design than the target gene itself.

One-step and sequential SARSCOV-2 polymerase chain reaction tests would not work every time.

INTRODUCTION: RT-PCR is widely used as a diagnostic test for the detection of SARS-CoV-2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID-19. METHOD: We collected multiple nasopharyngeal swab samples from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization. RESULT: Among 68 patients with initial negative test results, at the end of follow-up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. CONCLUSION: Based on this study, serial RT-PCR testing is unlikely to yield additional information.

Saving lives through early diagnosis: the promise and role of point of care testing for sickle cell disease.

Sickle cell disease (SCD) is a devastating and under-recognised global child health issue affecting over 300,000 infants annually, with the highest prevalence in India and sub-Saharan Africa. Most affected infants born in low- and middle-income countries (LMIC) lack access to SCD testing and die from complications in the first years of life without a formal diagnosis. The majority of deaths are preventable with early diagnosis and provision of inexpensive interventions. Despite global recognition of the urgent need, expansion of SCD newborn screening (NBS) programmes beyond the pilot stage has been obstructed by a dependence on an expensive and logistically challenging centralised laboratory testing model. Recently, several point-of-care tests (POCT) for SCD have been developed with promising field validation studies. Here, we summarise the state of POCT for SCD, review barriers and unanswered questions, and discuss optimal strategies for utilising POCT to address the growing global burden of SCD. There is an urgent need to prospectively evaluate the ability of POCT to reduce the morbidity and high early mortality of SCD. To impact a sustainable reduction to this end, it is essential to link a diagnosis with comprehensive SCD care, including wide and affordable access to affordable hydroxycarbamide therapy.

Confirmatory testing of Neisseria gonorrhoeae in a sexual health clinic: implications for epidemiology and treatment policy.

OBJECTIVES: European guidelines advise the use of dual nucleic acid amplification tests (NAAT) in order to minimise the inappropriate diagnosis of Neisseria gonorrhoeae (Ng) in urogenital samples from low prevalence areas and in extragenital specimens. In this cross-sectional study, we investigated the effect of confirmatory testing and confirmation policy on the Ng-positivity in a population visiting the sexual health clinic in Rotterdam, the Netherlands. METHODS: Apart from urogenital testing, extragenital (oropharyngeal/anorectal) testing was performed for men who have sex with men (MSM) and according to sexual exposure for women and heterosexual men. Ng detection using NAAT was performed using BD Viper and for confirmatory testing BD MAX. Sexual transmitted infection consultation data were merged with diagnostic data from August 2015 through May 2016. RESULTS: In women (n=4175), oral testing was performed in 84% and 22% were tested anally. In MSM (n=1828), these percentages were 97% and 96%, respectively. Heterosexual men (n=3089) were tested urogenitally. After confirmatory testing, oropharyngeal positivity rates decreased from 7.3% (95% CI 6.5 to 8.2) to 1.5% (95% CI 1.1 to 1.8) in women and from 13.9% (95% CI 12.3 to 15.5) to 5.4% (95% CI 4.3 to 6.4) in MSM. Anorectal positivity rates decreased from 2.6% (95% CI 1.6 to 3.7) to 1.8% (95% CI 0.9 to 2.6) in women and from 9.3% (95% CI 7.9 to 10.7) to 7.2% (95% CI 6.0 to 8.5) in MSM. Urogenital Ng-positivity rate ranged between 3.0% and 4.4% and after confirmation between 2.3% and 3.9%. When confirming oropharyngeal samples, Ng-positivity was 3.8% in women, 3.0% in heterosexual men and 12.5% in MSM. Additional confirmation of urogenital and anorectal samples led to 3.0% Ng positivity in women, 2.7% in heterosexual men and 11.4% in MSM. CONCLUSIONS: Confirmation of urogenital and anorectal samples reduced the Ng-positivity rates, especially for women. However, as there is no gold standard for the confirmation of Ng infection, the dilemma within public health settings is to choose between two evils: missing diagnoses or overtreatment. In view of the large decrease in oropharyngeal positivity, confirmation Ng-positivity in oropharyngeal samples remains essential to avoid unnecessary treatment.

Assessing an Adaptation of the Universal Parasite Diagnostic Assay for Bloodborne Parasites in a US State Public Health Laboratory.

For complex clinical cases where a parasitic infection is suspected, it can be difficult for clinicians to recommend an appropriate laboratory test. These tests are usually pathogen-specific and require a certain degree of suspicion for the precise etiology. A recently described assay, the universal parasite diagnostic (UPDx) can potentially provide a diagnosis of any parasite present in a specimen. Using primers that amplify DNA from all eukaryotes, UPDx differentiates several parasitic infections in blood by amplicon-based next-generation sequencing (NGS) of the 18S rDNA locus. As the state's public health reference laboratory, the Parasitology Laboratory at the Wadsworth Center (Albany, NY) receives specimens from patients who have potentially encountered a wide variety of parasites. As such, the ability to differentiate several blood parasites using a single assay is of interest. We assessed UPDx for its ability to confirm parasitic infections for 20 specimens that were previously identified by real-time PCR (RT-PCR). This included specimens positive for Babesia microti, Trypanosoma cruzi, Leishmania tropica, various Plasmodium species, and specimens comprising mixed Plasmodium sp. infections. Results obtained using UPDx were largely concordant with the RT-PCR assays. A T. cruzi positive specimen was negative by UPDx and for two mixed Plasmodium sp. infections only one species was detected. The results obtained for other specimens were concordant. We conclude that UPDx shows promise for the detection of blood parasites in diagnostic laboratories. As NGS becomes cheaper, assays like UPDx will become increasingly amenable to use in clinical settings.

Improved Detectability of Plasmodium falciparum Clones with Repeated Sampling in Incident and Chronic Infections in Burkina Faso.

We evaluated the detectability of Plasmodium falciparum clones when assessed on 3 consecutive days in incident and chronic infections in naturally exposed children living in an area of intense malaria transmission in Burkina Faso. The median number of clones by merozoite surface protein 2 (MSP2) genotyping was 3 (interquartile range [IQR] 2-5) in incident infections compared with 6 (IQR 4-8) in chronic infections (P < 0.0001). When all clones detected on days 1-3 were considered as true complexity of infection, sampling on day 1 detected only 69.4% (109/157) or 68.3% (228/334) of all clones in incident and chronic infections, respectively. Our findings demonstrate that a large proportion of clones are missed by single time-point sampling. In addition, because of the high complexity of infection early in incident infections, our data suggest many infections may be caused by genetically complex inocula.

Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification.

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.