Point-of-care (POC) molecular diagnostics for clinical microbiology and virology has primarily focused on the detection of a single pathogen. More recently, it has transitioned into a comprehensive syndromic approach that employs multiplex capabilities, including the simultaneous detection of two or more pathogens. Multiplex POC tests provide higher accuracy to for actionable decisionmaking in critical care, which leads to pathogen-specific treatment and standardized usages of antibiotics that help prevent unnecessary processes. In addition, these tests can be simple enough to operate at the primary care level and in remote settings where there is no laboratory infrastructure. This review focuses on state-of-the-art multiplexed molecular point-of-care tests (POCT) for infectious diseases and efforts to overcome their limitations, especially related to inadequate throughput for the identification of syndromic diseases. We also discuss promising and imperative clinical POC approaches, as well as the possible hurdles of their practical applications as front-line diagnostic tests. In fact, the treatment of infectious diseases are rarely considered to be model applications of personalized medicine; however, this perception is gradually changing due to a substantial increase in antimicrobial resistance (AMR) to antibiotics due to unnecessary usage [A personalized medicine and treatment regimen, which establishes clinical plans on a patient-by-patient basis in the treatment of infectious diseases, is a trending topic in the field of clinical microbiology and virology –5. In gery usage –9. Sincery usage –12. The ry usage –17. In ary usage , 17, 18.S. aureus, C. difficile, enterococci or multi-resistant enterobacteria [Clinical microbiological and virological POC molecular diagnostics have initially addressed the optimization of technologies from centralized laboratories to the medical checkup field, regardless of the number of detectable pathogens . The fiebacteria . Howeverbacteria . Infectibacteria , 24. InfThis review focuses on up-to-date progress in multiplexed molecular POCT for infectious diseases and efforts to overcome limitations. We have summarized information on recently approved devices and state-of-the-art developments through a comprehensive literature review. We also discuss the potential clinical impact of the recent POC approach and the possible hurdles that impede their practical application as a leading diagnostic test.C. difficile, or methicillin-resistant Staphylococcus aureus (MRSA). The first molecular test designed for POC was approved by the U.S. Food and Drug Administration (FDA) in 2015. ID NOW™ Influenza A & B 2, formerly known as Alere I influenza A & B 2, , provides highly sensitive results in 13 min or less. It is based on isothermal nucleic acid amplification technology, which uses nicking enzyme amplification reaction (NEAR) technology. As many emerging isothermal amplified POC applications have reached the proof-of-concept stage, ID NOW™ has successfully entered the infectious disease POC market for influenza A & B, Strep A and respiratory syncytial virus (RSV) testing [® Liat influenza A/B/RSV assays  and GeneXpert® Xpress Flu/RSV  are known as Clinical Laboratory Improvement Amendments (CLIA)-waived (CW) assays that enable non-training personnel to obtain results within 30–50 min and are suitable for POCT [® Xpress Flu/RSV are based on real-time reverse transcription-polymerase chain reaction (rtRT-PCR), which can detect both influenza A/B and RSV during a single run [io , which combines PCR amplification and electrochemical detection technology, has recently received 510(k) clearance from the U.S. FDA for the dual targets of chlamydia (CT) and gonorrhea (NG) [®  exemplifies this idea of a technical breakthrough enabling simultaneous detection of multiple targets. The microfluidic pouch system connects to a module for DNA extraction and purification and employs a post-PCR melting curve analysis that differentiates targets based on the distinct melting temperature (Tm) peaks for each target. This allows for the multiplexed detection of multiple targets. Another example is the FDA-cleared Verigene  system, which that uses gold nanoparticle probes and a microarray for the detection of bacterial pathogens and several resistance markers. However, neither platform is classified as a POC test.The implementation of NAATs has been a huge leap forward in test sensitivity and time-to-results. There are several molecular-based assays that meet the criteria for POC application and have been approved or are clinically available for the detection of single-key pathogens, such as influenza,  testing –27. ID Nfor POCT . Howeverngle run –31. The hea (NG) , 33. Thehea (NG) . Moreovehea (NG) . The novStaphylococcus aureus DNA from human blood samples in 30 min with separation, amplification, and digital quantitative nucleic acid readouts. The result simplifies molecular testing, making it portable, and available for rapid medical decisionmaking. On the other hand, NAATs that utilize paper substrate matrices provide a low-cost, easy-to-store, and portable platform. Klapperich’s et al. developed a single paper-fluidic-chip-based lateral flow assay, which allows for the completion of NAAT steps from sample to result [Microfluidic devices for molecular diagnostics have been less commercialized over the last few years compared to the immense academic interest focusing on the miniaturization of molecular biological techniques, including target gene amplification onto microfluidic devices. However, there is an increasing need to integrate microfluidic devices with other applications, such as sample pre-treatment, fluid control, and multiplex capability Fig. . For exao result . The devSalmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7, and a multi-drug-resistant bacteria Acinetobacter baumannii [Numerous studies have highlighted the importance of a syndromic approach in a multiplex molecular diagnostic test –41. A syaumannii . The plan 45 min . This as®) signal, which is caused by hybridization of the PCR-amplified pathogen DNA and the capture of probe-decorated nanoparticles [Candida species (which account for 95% of candidemia) but is able to provide antifungal susceptibility data [®) is a multiplex molecular diagnostic system that enables fast, cost-effective, and flexible syndromic testing. The system has received CE-IVD certification for a respiratory panel that detects 21 pathogens and will be able to process up to 48 molecular targets at once, which will permit the diagnosis of serious respiratory and gastrointestinal infections.Numerous studies involving multiplex molecular POC are now moving toward extensive diagnosis capability, but few kits have been approved for the detection of multiple pathogens based on clinical syndromes Table . For exaarticles . The T2 ity data . Due to A few years ago, molecular POC diagnosis was limited to testing only the most common pathogens. This lack in diagnostics capability led to further downstream tests or unnecessary prescription of antibiotics. The recently commercialized multiplex molecular assays enable simultaneous detection and identification of multiple pathogens associated with clinical syndromes with regard to bloodstream, respiratory, gastrointestinal (GI), or central nervous system infections . As the e.g., lateral flow, are commonly known to lack sensitivity. Those established tests could be replaced by rapid molecular diagnostics that are simple, easy to use, and highly accurate compared to the former gold standard methods. In this review, we focused on recent progress in molecular POC testing for infectious diseases and summarize the available information regarding recently approved devices and state-of-the-art developments. The implementation of multiplex molecular POC testing will result in unprecedented changes in antimicrobial therapeutic decisions, management, and control of infectious diseases. Despite technological advances, there are still a great deal of challenges for multiplex molecular POC tests in clinical practice, such as moderate test performance, complexity that make it difficult to use in many clinical settings, limited access to testing, and high costs. Considering that countless researchers have encouraged finding a way to overcome these challenges, we believe that multiplex molecular POC will become a powerful principal diagnostic test.Evolving POC molecular diagnostics are closer to realization in the medical field. The successful migration of a molecular POC test has changed the trends of centralized laboratory testing from general molecular diagnostics with highly skilled experts and complex equipment to the more comprehensive molecular diagnostics in a clinical setting using simpler instruments. The emergence of a new CLIA-waived molecular POC platform has received major attention in the global in vitro diagnostics (IVD) market, because traditional POC tests,


Delay in closure of ductus arteriosus in postnatal life may lead to serious consequences and complications in an extremely premature neonate secondary to hemodynamic alterations in regional blood flow pattern in various organs. Despite the widespread recognition amongst neonatologists to identify a hemodynamically significant patent ductus arteriosus (hsPDA) early in the postnatal course, there is lack of consensus in its definition and thus the threshold to initiate treatment. Echocardiographic assessment of PDA shunt size and volume combined with neonatologists' impression of clinical significance is most frequently used to determine the need for treatment of PDA. Common clinical signs of hsPDA utilized as surrogate for decreased tissue perfusion may lag behind early echocardiographic signs. Although echocardiogram allows direct assessment of PDA shunt and hemodynamic alterations in the heart, it is limited by dependence on pediatric cardiologist availability, interobserver variation and isolated time point assessment. Electrical cardiometry (EC) is a non-invasive continuous real time measurement of cardiac output by applying changes in thoracic electrical impedance. EC has been validated in preterm newborns by concomitant transthoracic echocardiogram assessments and may be beneficial in studying changes in cardiac output in premature newborns with hsPDA. Alterations in perfusion index derived from continuous pulse oximetry monitoring has been used to study changes in cardiac performance and tissue perfusion in infants with PDA. Near infrared spectroscopy (NIRS) has been used to objectively and continuously assess variations in renal, mesenteric, and cerebral oxygen saturation and thus perfusion changes due to diastolic vascular steal from hsPDA in preterm neonates. Doppler ultrasound studies measuring resistive indices in cerebral circulation indicate disturbance in cerebral perfusion secondary to ductal steal. With recent trends of change in practice toward less intervention in care of preterm newborn, treatment strategy needs to be targeted for select preterm population most vulnerable to adverse hemodynamic effects of PDA. Integration of these novel ways of hemodynamic and tissue perfusion assessment in routine clinical care may help mitigate the challenges in defining and targeting treatment of hsPDA thereby improving outcomes in extremely premature neonates. Ductus arteriosus is an important and necessary structure for the fetus. It allows communication between the pulmonary artery and descending aorta during fetal life facilitating majority of right ventricular output to bypass the pulmonary vascular bed and enter the descending aorta supporting systemic oxygenation. Delay in its postnatal closure as commonly encountered in extremely premature infants born <28 weeks gestation  can resuThe clinical consequences depend on the degree of left-to-right shunting through the PDA and ductal steal. The increase in pulmonary blood flow in the setting of prematurity can lead to pulmonary edema, pulmonary hemorrhage, respiratory deterioration, pulmonary hypertension, left atrial and ventricular overload and dilation followed by left ventricular dysfunction , 4. DimiPersistent patency of hsPDA is indisputably associated with increased morbidity and mortality in premature neonates. However, there is debate whether early medical or surgical closure of PDA improves outcomes. Hence, in the last decade, approach to PDA has shifted from early treatment to watchful waiting for spontaneous closure . A recenCurrently echocardiographic measurements of a PDA are considered as gold standard for assessing the magnitude and relevance of left to right shunting in premature newborns . Many auArterial blood pressure (BP) is one of the commonly used physiologic signals for evaluating hemodynamic status associated with any clinical condition. Invasive arterial catheter is the gold standard for measuring arterial BP in the initial course of premature newborn followed by oscillometric monitoring. There are technical concerns with inaccurate and under or over estimation of BP with either methods in this population , 18 howeEchocardiography is the gold standard of predicting and diagnosing hemodynamic significance of PDA. hsPDA may go clinically unnoticed in initial postnatal course of a preterm neonate as clinicals signs of hsPDA lag behind echocardiographic measurements by mean of 2 days . PerformThe European special interest group for Neonatologist Performed Echocardiography  describeVarious scoring systems, incorporating measurements from fECHO have been published to standardize the assessment of hemodynamic severity of the PDA , 38. TheAssessment of the hemodynamic status particularly cardiac function is crucial for understanding the hemodynamic impact of hsPDA in preterm neonates. In neonatal practice, invasive blood pressure is routinely and continuously monitored in initial days of life for premature infants followed by intermittent non-invasive oscillometric blood pressure monitoring. Assessment of cardiac output (CO) by interpretation of indirect variables like blood pressure can be unreliable in premature and sick neonates . Other rElectrical Cardiometry  has been proposed as a safe, well-tolerated, practical, non-invasive, continuous method of measurement of cardiac output that is reproducible in neonates of various gestational maturity, weight and body surface area , 47. EC Authors have established the validity of EC by demonstrating that stroke volume and cardiac output measured by EC are comparable with echocardiogram with estimated similar bias, precision and acceptable percentage error in neonates , 54, 57.Despite the accumulating evidence of clinically advantageous and acceptable values between EC and echocardiogram in hemodynamic assessment of hsPDA, it is important to note that reference values of premature neonate cardiac function on EC is yet under research and not clinically established . Also la2) in multiple organs particularly cerebral (cRSO2), renal (rRSO2), and splanchnic beds (sRSO2) necessitating physiological increase in fractional tissue oxygen extraction (FTOE) to maintain tissue oxygen requirement in premature neonates. Use of NIRS has progressed from being a research tool to clinical bedside device for non-invasive, continuous assessment of this changing oxygen dynamics in presence of hsPDA  is a non-invasive method of objectively measuring peripheral perfusion from the plethysmography signal generated by pulse oximeters and represents the ratio of light absorbed by pulsatile elements such as arteries and non-pulsatile elements such as blood in venous or capillary bed, bone, connective tissue etc. , 84. PI Studies have examined PI values in healthy and sick term neonates  as well Decrease in superior vena cava (SVC) flow may be a novel marker of compromised systemic circulation in neonates with hsPDA . PI has Other authors reported that the PI of the lower extremities  is decreased compared to the right arm  in preterm infants with hsPDA which measured by delta PI of a certain cut off value strongly correlated with the echocardiographic diagnosis of hsPDA . In anotThe review of physiologic alterations secondary to hsPDA would be incomplete without discussion of certain biomarkers that have been studied and incorporated in clinical practice for assessment of hemodynamic significance of PDA. Cardiac peptides such as B-type (Brain) natriuretic peptide (BNP) and N-terminal pro-BNP(NT-proBNP) have been studied extensively for early detection as well as monitoring therapy of hsPDA. NT-pro BNP is a marker of heart failure that increases due to ventricular volume and pressure overload in hsPDA. Normative values for this peptide in plasma in first week of life has been studied at various gestational ages, with lower normal values noted in term infants compared to preterms . VariousAlterations of organ perfusion associated with hsPDA can lead to production of ischemia modified albumin, serum levels of which were found to be elevated in cohort of preterm neonates with hsPDA . RelatioElevated nucleated red blood cell (NRBC) count at birth is related to chronic uterine hypoxia which may predispose to PDA. A recent prospective study of preterm infants, showed differences in absolute NRBC count with ductal size and hemodynamic significance of PDA as measured by echocardiogram. The authors proposed cut off absolute NRBC levels to predict hsPDA .There is ambiguous data on association of thrombocytopenia with hsPDA and its influence on response to treatment. Presence of low platelet count and high platelet distribution width in preterm neonates is shown to be associated with hsPDA . There iThe definition of hsPDA and the population it applies to, continues to develop and unfold with accumulation of further evidence of novel ways of assessment and outcomes. This review discusses the role of various continuous modes of tapping and analyzing physiologic variables resulting from a hsPDA beyond the cardiologist's echocardiogram as summarized in AP conceived the idea for the article, reviewed the current literature, and wrote the manuscript. PT reviewed the literature and assisted in writing various sections of the manuscript. MM and HB critically reviewed the manuscript. All authors reviewed the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To raise awareness of complement factor I (CFI) deficiency as a potentially treatable cause of severe cerebral inflammation.Case report with neuroradiology, neuropathology, and functional data describing the mutation with review of literature.We present a case of acute, fulminant, destructive cerebral edema in a previously well 11-year-old, demonstrating massive activation of complement pathways on neuropathology and compound heterozygote status for 2 pathogenic mutations in CFI which result in normal levels but completely abrogate function.Our case adds to a very small number of extant reports of this phenomenon associated with a spectrum of inflammatory histopathologies including hemorrhagic leukoencephalopathy and clinical presentations resembling severe acute disseminated encephalomyelitis. CFI deficiency can result in uncontrolled activation of the complement pathways in the brain resulting in devastating cerebral inflammation. The deficit is latent, but the catastrophic dysregulation of the complement system may be the result of a C3 acute phase response. Diagnoses to date have been retrospective. Diagnosis requires a high index of suspicion and clinician awareness of the limitations of first-line clinical tests of complement activity and activation. Simple measurement of circulating CFI levels, as here, may fail to diagnose functional deficiency with absent CFI activity. These diagnostic challenges may mean that the CFI deficiency is being systematically under-recognized as a cause of fulminant cerebral inflammation. Complement inhibitory therapies (such as eculizumab) offer new potential treatment, underlining the importance of prompt recognition, and real-time whole exome sequencing may play an important future role. CFI deficiency may be an under-recognized cause of encephalitis of “presumed viral” or unknown etiology. Improved outcome will require greater awareness of the condition and a high index of suspicion.We report a case of life-threatening, nonhemorrhagic fulminant CNS inflammation, radiologically resembling acute disseminated encephalomyelitis (ADEM), in association with complete complement factor I (CFI) functional deficiency. A very few such cases have been reported to date, all identified retrospectively via whole exome sequencing (WES) and/or known family history. Complement inhibition  represents a potential therapeutic option in this otherwise devastating illness but would require prompt recognition. The index case had a functional CFI deficiency  for a working diagnosis of severe ADEM. There was little obvious benefit. The severely elevated intracranial pressure began to settle around day 6. By day 14, some withdrawal from painful stimuli was noted. Blood and CSF cultures were negative for primary bacterial infection. CSF virology was negative for herpes simplex, varicella, Enterovirus, and Parechovirus by PCR. An initial CSF sample was heavily blood-stained with protein 0.81g/L and 30,000 erythrocytes/mm3 but no excess of CSF leukocytes. Subsequent CSF samples showed no CSF leukocytes. Antibodies to myelin-oligodendrocyte glycoprotein, aquaporin 4, glycine receptors, voltage-gated potassium channel-related proteins, and NMDA receptors were all negative. Immunoglobulin levels at presentation were normal.She was treated with ceftriaxone and aciclovir for presumed meningoencephalitis, high-dose methylprednisolone (1 g daily for 5 days), followed by an extended high-dose enteral prednisolone taper; plasmapheresis with human albumin solution and fresh frozen plasma (FFP) (days 5–15 inclusive); and rituximab  and c.1555 G>A p.(Asp519Asn). Subsequent Sanger sequencing of the proband and parents confirmed compound heterozygote status. Subsequent complement assays were performed 8 months after presentation. C3 and C4 were within the normal range  0.68–1.80] and 0.35 [NR 0.18–0.6] g/L, respectively). CFI and complement factor H (CFH) were within the normal range . There was marked activation of the alternative pathway (AP) with markedly low complement factor B  and absent AP haemolytic activity (AP100).links.lww.com/NXI/A208).Nearly 2 years from presentation, the patient has been left with a profound neurologic deficit with tetraplegia, although she can write slowly with specialist aids indicating relative cognitive preservation. A seizure disorder is well controlled with levetiracetam. She has commenced penicillin prophylaxis and has been immunized against capsulated organisms in light of the absent AP activity. Follow-up imaging at 6 months showed severe widespread atrophy with no new signal abnormality  2 and age-related macular degeneration.3 CFI-mediated complement regulation may also be important in the progression of early phase Alzheimer disease.4We describe a patient with compound heterozygote, complete CFI deficiency, with a nonclassic, neurologic presentation. CFI is a complement regulatory protein that inactivates all pathways of the complement system by cleaving the α′-chains of C3b and C4b . Complet5 and a recent adult brief report6 describe acute hemorrhagic leukoencephalopathy (AHLE) in association with complete CFI deficiency. All had infrequently recurrent episodes, at intervals of several years, commencing in the second decade of life. Radiologically, our case resembles case B in Shields et al.,6 but appears uniquely severely affected among cases reported to date.A report of 2 unrelated children of Filipino descentWe speculate that the basis for such infrequent, fulminant presentations may be that the CFI deficiency causes chronic AP activation limited only by depletion of its substrate C3. C3 is an acute phase protein. and stressors such as viral infection may result in a rapid surge in C3 production, freeing the previously substrate-limited AP and resulting in a catastrophic runaway that overwhelms downstream complement regulators (which are in any case poorly expressed in the brain). In this context, one could hypothesize that our use of plasmapheresis with FFP may have been deleterious because of delivery of exogenous  factor I and C3, facilitating proinflammatory conversion of C3b to iC3b , althoug8 suggesting a basis for similar radiologic appearances.Our case had no radiologic or pathologic evidence of hemorrhage, suggesting an AHLE phenotype is not consistent and that neuroradiologic appearances can resemble acute demyelination. Complement activation is a recognized pathophysiologic mechanism in neuromyelitis optica and related disorders,links.lww.com/NXI/A208). In both instances, absolute levels of CFI were normal; however, functional analysis demonstrated almost complete abrogation of enzymatic activity.9 Thus, unlike previous cases,5 our patient was in the highly unusual situation of having antigenically normal CFI levels but severely deficient CFI activity from both mutant alleles. In addition, the unexpected finding of normal serum C3 level in the serum reflects the fact that routine clinical assays may also detect other forms of C3, in this setting, most likely C3b. The most reliable routinely clinically available diagnostic clue in this situation is the markedly reduced CFB level that reflects excessive CFB consumption because of a lack of inhibition of the amplification loop  in association with aHUS . Real-time WES10 may also play an important future role in diagnosis. The identification of complement regulator defects would provide a biological rational for the use of eculizumab and the next wave of 2nd generation complement inhibitory therapies.In keeping with other reports,Deidentified clinical data will be made available on appropriate request to the corresponding author.


Routine microbiology results are a valuable source of antimicrobial resistance (AMR) surveillance data in low- and middle-income countries (LMICs) as well as in high-income countries. Different approaches and strategies are used to generate AMR surveillance data.We aimed to review strategies for AMR surveillance using routine microbiology results in LMICs and to highlight areas that need support to generate high-quality AMR data.We searched PubMed for papers that used routine microbiology to describe the epidemiology of AMR and drug-resistant infections in LMICs. We also included papers that, from our perspective, were critical in highlighting the biases and challenges or employed specific strategies to overcome these in reporting AMR surveillance in LMICs.Topics covered included strategies of identifying AMR cases , of collecting data , of sampling AMR cases , and of processing and analysing data for AMR surveillance in LMICs.The various AMR surveillance strategies warrant a thorough understanding of their limitations and potential biases to ensure maximum utilization and interpretation of local routine microbiology data across time and space. For instance, surveillance using case-finding based on results from clinical diagnostic specimens is relatively easy to implement and sustain in LMIC settings, but the estimates of incidence and proportion of AMR is at risk of biases due to underuse of microbiology. Casebased surveillance of clinical syndromes generates informative statistics that can be translated to clinical practices but needs financial and technical support as well as locally tailored trainings to sustain. Innovative AMR surveillance strategies that can easily be implemented and sustained with minimal costs will be useful for improving AMR data availability and quality in LMICs. Surveillance of antimicrobial resistance (AMR) is a fundamental action to inform efforts to control the spread of AMR through generation of evidence for local, national and global guidance and action plans . The maiVarious AMR surveillance modalities have been described by the World Health Organization (WHO) . SurveilAt sentinel sites, AMR surveillance involves collection, validation, interpretation and reporting of susceptibility data. Microbiological testing is a crucial tool for diagnosis of bacterial infections and a source of AMR data in LMICs. Optimizing existing tools for diagnosing bacterial infections and generating bacterial identification and antimicrobial susceptibility data on site is the foundation of an AMR surveillance system ,9. PotenPubMed was searched for literature published before 1st November 2020, with the main theme of AMR surveillance using routine microbiology data in LMICs as search terms. Titles, abstracts, and full texts of search results were reviewed see . MoreoveThere is an extensive body of literature on different strategies for identifying AMR/DRIs in a prespecified population using routine microbiology data ,13. In gSalmonella spp [This approach was described in the WHO Global AMR Surveillance System (GLASS): cases are identified through microbiology results from routinely collected clinical diagnostic specimens . This meella spp . Alternaella spp ,19—22. Aella spp , and theella spp ; thus usella spp ,24.A potential concern of AMR surveillance based on routine microbiology data alone is the representativeness of data for the target population, especially when microbiological diagnosis is not performed systematically and important parameters are not captured. First, diagnostic microbiology testing may be systematically underused (i.e. patients with infectious diseases are treated with empiric antibiotics without blood culture testing) and selectively delayed  in LMICs ,26. ConsThis approach is based on patients who present for medical care with signs and symptoms of infection. Compared to the laboratory-based approach, the case-based approach is more robust to variations in the use of microbiology and selection bias, as case identification and specimen collection time is based on more systematic and objective data collection ,14. MoreA key advantage of case-based surveillance is that the data can be used to support clinical decision-making and choice of empiric antibiotics ,33,34. TIncreasingly, case-based surveillance has been discussed ,8,13 andThere are challenges in implementing case-based surveillance in LMICs. First, comprehensive clinical data are not available in electronic format in most LMIC settings. Clinical data are important for defining infections, but routine documentation of such data and the use of accepted case definitions can be challenging in LMICs. The challenges include a low ratio of healthcare-workers to patients, lack of supporting tests (e.g. radiology), and lack of diagnostic guidelines to rapidly differentiate bacterial from other infections. Second, there is a lack of trained personnel and guidelines in local languages to support case-based surveillance. Third, there is a lack of open-access, easy-to-use information technology to support clinical data capturing and storage and often limited exchange of clinical information between clinicians and the laboratory which may impact the quality/relevance of laboratory results . Fourth,Multiple survey designs have been used to collect AMR data in hospitals in LMICs. These include cross-sectional point-prevalence surveys (PPSs), observational cohorts, and case—control designs. PPSs have been used to measure the prevalence of healthcare-associated infections and antimicrobial use and resistance in healthcare settings in LMICs ,39,40. IWhen population-based surveillance cannot be performed for practical reasons, various sampling approaches can be considered. Randomly sampled study cohorts and/or surveillance sites from a large target population is a common approach. Studies using lot quality assurance sampling (LQAS) have been shown to be useful in informing whether or not resistance of a pathogen to specific antibiotics is high, which is essential to inform the appropriateness of using those antibiotics for empiric treatment ,51. LQASThe considerations for when sampling needs to be done are the sample size, sampling approach, and sampling frequency . The keyWhen attempting to estimate the health impact due to AMR infections, various sampling strategies for comparator cohorts have been suggested. WHO GLASS recently published a guideline for estimating attributable mortality of AMR bloodstream infections (BSIs) , in whicAt the global level, GLASS published protocols to standardize AMR surveillance data processing. In general, it is recommended that repeated isolates from individual patients are to be removed before estimating prevalence and incidence of AMR infections (deduplication), and data should be stratified by infection origin. Increasingly, open-access applications such as WHONET  and AMASAt the national and global level, population catchment and pattern of healthcare utilization in the population are ideally to be included in AMR surveillance reports to interpret AMR surveillance data. However, such statistics are not commonly available in LMICs. The use of data from existing Health and Demographic Surveillance Sites (HDSS) to combine community- and hospital-based clinical and microbiology data has been recommended ,57 but hA paradigm shift in the value of microbiological testing and prioritization of the detection of drug-resistant infections is needed in many LMICs . InnovatWhile the use of microbiology in many LMICs is gradually increasing, essential parameters such as blood culture use rate and rates of AMR stratified by antibiotic usage should also be used to standardize the measures of prevalence and incidence, and increase the comparability of AMR burden across space and time .Social and behavioural changes on the attitudes and practices of antibiotic usage and microbiological testing are needed and may be accelerated with diagnostic and antibiotic stewardship programmes. Systematic trainings to increase awareness on the significance of microbiology testing among all stakeholders, from students to senior specialists, may motivate the use of microbiology.Investments in improving microbiology quality and use through expensive automated equipment do not always deliver, as operating costs and costs of maintenance and consumables have not been taken into account. Maintaining power supply and cold chain often remains challenging. Innovative tools to simplify microbiological diagnosis  and to cThe incremental value of the case-based approach can be significant if implementation challenges can be overcome . TraininQuality assurance and control (QA/QC) are essential to ensure accurate identification of pathogens and AST profiles, and need to be improved in sentinel surveillance sites in LMICs. Commercial QA/QC schemes and accreditation programmes are available, but often unaffordable for laboratories in LMICs. Training and support to ensure quality performance and methods of bacterial identification and AST are needed for laboratories in LMICs . Web-basVerifying AST results of bacterial isolates is an important component of surveillance to ensure quality data are generated. However, data verification and highlighting isolates with unusual AST results can be a complicated and time-consuming process to perform manually. Commercial laboratory information management systems with functions to support microbiology data verification are unaffordable for many hospitals in LMICs. The openaccess WHONET programme has functions to support data quality checks . Open-acFinally, there is a lack of evaluation frameworks for continuous AMR surveillance systems in LMICs. Regular evaluations on the performance of AMR surveillance systems in LMICs would be useful to identify limitations and areas for improvement as well as to quantify and qualify progress when it occurs. Examples of evaluation tools for AMR surveillance network are the AMR Progressive Management Pathway tool developed by the Food and Agriculture Organization of the United Nations, NEOH and SURV-TOOLS . The impSeveral strategies for conducting AMR surveillance have been used in LMICs, with the majority adopting the isolate- or samplebased approach using microbiology data from routine clinical diagnostics. Attempts to transition to a case-based approach are receiving increased attention, but significant challenges remain in settings where resources, expertise and experience are limited. Guidelines, trainings, and local capacity building are useful to support such transitions. Moreover, tertiary hospitals, research institutions, and pharmaceutical companies with microbiology testing and involved in AMR data collection could play a role in leading and supporting the transition to case-based AMR surveillance in surrounding facilities within their reach. In parallel, it is important to strengthen diagnostic capacity, reduce costs and otherwise incentivize routine microbiology testing, especially when most surveillance still relies on a laboratory-based approach in central reference hospitals. Strategies to maximize usage of locally existing resources and data could narrow the data gap. Improvements in AMR data generated from LMICs also depends on innovative and collaborative clinical research on strategies that can require minimal financial and human resource requirements while data quality is maintained. For instance, the LQAS has shown applicability in LMICs to inform empirical antibiotic treatment guidelines in a local setting, and WISCA could be an informative strategy for presenting AMR patterns to guide treatment decisions. Experience from ACORN implementation and further roll-out will inform what is needed for this transition in LMICs. Finally, innovative AMR surveillance tools and strategies that can be easily implemented and maintained in resource-limited settings will be useful for improving AMR data availability and quality to support local, national and global action plans in controlling AMR.https://doi.org/10.1016/j.cmi.2021.05.037.Supplementary data to this article can be found online at Supplementary data


Bovine tuberculosis (bTB) remains a major public health issue in Zambia and has been exacerbated by human immunodeficiency virus prevalence and consumption of unpasteurized milk in the Southern Province of the country. The prevalence of bTB has been established to be linked to Kafue Lechwe, which act as reservoir hosts and share grazing fields with domestic cattle. No studies have so far used geographic information system (GIS) to investigate the relationship between the reservoir hosts (Kafue Lechwe) and domestic animals. This study, therefore, aimed to apply GIS to investigate the spatial distribution of bTB in Namwala District of the Southern Province of the country.To investigate the spatial distribution of bTB, geographical positioning system (GPS) coordinates representing 96 cattle herds across 20 independent villages were captured alongside risk factor data. The 96 herds were based on abattoir reports of condemned carcasses and a trace back. Positive herds were confirmed by cross-reference to purified protein derivative tests conducted by the District Veterinary Office. The GPS coordinates were transferred into ArcView 3.2 and laid on the map of Namwala District alongside physical features, including national parks, game management areas, and flood plains. Questionnaires were administered across 96 independent households to assess risk factors of bTB transmission.The results revealed a “clustered” spatial distribution of the disease in cattle in Namwala District of Zambia, particularly significant in the eastern interface areas of the district (p=0.006 using Moran’s I). Abattoir to production area trace back revealed a herd-level prevalence of 36.4% (95% CI=26.7-46.3%) among cattle herds in Namwala District, whereas individual animal prevalence ranged from 0% to 14% (95% CI=2.4-26.2%). Further, GPS data indicated that the majority of the positive herds were located at the livestock/wildlife interface area. Contacts with wildlife, coupled with sharing grazing, and watering points were found to be significant risk factors for bTB transmission.This study demonstrated the presence of bTB in cattle and associated spatial risk factors. In particular, bTB was observed to be a function of animal location within the livestock/wildlife interface area. GIS is thus an applicable and important tool in studying disease distribution. Mycobacterium bovis is a major zoonotic disease of worldwide economic and public health importance, especially in developing countries where control measures are neither formulated nor enforced [Mycobacterium tuberculosis complex (MTC). Notably, bTB has been shown to exhibit a wide host range and preference than most pathogens within the MTC, making it a very important emerging zoonotic pathogen [M. tuberculosis is the most common cause of human TB, but an unknown proportion of cases are due to M. bovis [M. bovis and M. tuberculosis originating types), implicating TB as the greatest cause of death due to a single pathogen, second only to the HIV [Bovine tuberculosis (bTB) caused by enforced ,2. In fapathogen . Among tM. bovis ,4. The rM. bovis ,2. Thus,M. bovis . The Wor the HIV -9. In so the HIV ,11. A co the HIV . However the HIV .Geovisualization or geographic information system (GIS) mapping is the use of computer-assisted graphical methods to visualize geospatial information. It is a method that is used to help guide and direct health service planning, public health interventions and to capture and reveal disease hotspots . The aimBased on the literature reviewed, a number of studies have been done on TB in Zambia. However, none has focused on spatial distribution, let alone the use of GIS tools in relation to risk factors. Such information would be vital to the management of the disease. If control programs were to be implemented with the available information, it may be difficult to know exactly where those efforts would have to be directed. The aim of this study was, therefore, to use GIS to help identify the spatial distribution of bTB in cattle of Namwala District of Zambia and identify spatially associated risk factors.The study did not require ethical approval as advised by the Department of Disease Control, School of Veterinary Medicine of the University of Zambia under which the study was conducted. However, informed consent was still obtained from each participant and confidentiality was observed.The study was conducted in Namwala District of Zambia located at 15°45’S and 26 °27’E .M. bovis. The study was conducted from October 2012 to March 2013 across different villages represented under the constituencies and cattle clubs therein  device and recorded.A closed-ended pre-tested structured questionnaire was used to collect data on age, sex, and other production factors of cattle reared. The biodata of the cattle rearing communities was also collected and accompanied with demographical and ecological data and from this information descriptive statistical tests were run.The collected coordinates were processed and transferred onto maps of Namwala to show bTB occurrence. The general pattern helped to determine how bTB distribution relates to the interface area. The processing and creation of maps were done in Arc-GIS 3.2. The points were first transferred onto a Microsoft Excel 2007 spreadsheet for cleaning and conversion to wholly decimal coordinates since the GPS device captured was in the form of ‘degrees’ and ‘minutes’. The cleaned data were stored as a text file, which were transferred into ArcGIS 3.2 to lay on the processed maps of Namwala District. The map layering included a combination of shapefiles to show important features of the district including most importantly, the flood plains, the rivers, game management areas (GMAs), and national parks. The captured coordinates were laid on the created maps to gain an understanding of how they relate to those important geographical features such as the flood plains, the GMAs, and national parks. The GMAs and national parks have been known to play a role in being the basis for the interface of wildlife and domestic animals where transmission of bTB has been known to occur. The analysis attempted to show how the coordinates indicating where animals found positive for TB originated related to the interface, which was a risk factor for the disease. While transmission occurs between livestock and wild animals, it does not always involve livestock wandering into GMA’s or national parks and neither does it always involve wild animals wandering far into grazing areas. Mixing tends to occur in areas that lie between either GMA’s or national parks and grazing area (interface). For our analysis, we posited buffer zones as interface areas where transmission of TB may occur from wild animals to livestock.Based on findings from a study in Nepal using GIS investigating buffer zones, 10-30 km buffers were created  from the® 2007 before transferring to Stata SE12 for Windows  for further analysis. The database included biodata, bTB test results as well as other epidemiological data. The proportion of bTB infected cattle in those slaughtered at the two slaughterhouses was estimated. Extraneous factors that could explain the infection of bTB in humans were described using both qualitative and analytical tools. The questionnaire was used to investigate possible factors that lead to domestic animals contracting bTB from reservoir hosts and possible practices leading to disease in humans. To assess the plausibility of the factors captured in the questionnaire, univariate analysis was used. Moreover, to understand the interaction of the factors observed on univariate analysis multiple regressions was used. Descriptive statistics were used to highlight characteristics of at-risk population and to help policy-makers in formulating control measures such as being able to know the most affected age group or sex group.The database establishment was in Microsoft ExcelUsing trace back system based on abattoir survey reports, a total of 96 herds of cattle from 20 different geo-captured villages in Namwala District were sampled for bTB positivity. The herd positivity was based on condemnation at the abattoirs and further information on relevant clinical signs gathered during interviews after tracing back to villages of origin of the individual animals condemned. Confirmation was from data records from animal health inspectors who carry out bTB testing using the purified protein derivative. From these, a total of 35 herds were found positive representing a herd-level prevalence of 36.4% (95% CI=26.7-46.3%) . IndividThe cluster of villages studied, grouped under one village name, had in the previous 12 months under study at least had one TB patient who had been treated or was receiving treatment for TB, according to the information gathered during the interviews. All these studied areas had their GPS location collected .The smaller circle within Majority of cattle herds that were found to be positive belonged to the villages in the eastern part of Namwala District 70% (n=14 villages), with 50% (n=10 villages) being located deep inside the buffer zone (the GMA) between the open areas and the National Park as indicated by the larger circle.There was an overall significant difference in bTB prevalence across the 20 villages studied based on individual animal data (p=0.001). Based on results for condemnation of carcasses, the individual herd prevalence of bTB ranged from 0 to 14%. In most areas, the prevalence oscillated around 3% .When GMAs were considered, the positive herds still tended to fall within the areas where mixing with wildlife occurs. The positive herds were within a 10 km buffer of the management areas .To test for correlation of positive bTB herds in the observed geographical areas, Moran’s I was used, which indicated a considerable correlation with p=0.006 . The corA total of 20 village clusters were studied from within the main villages, there were about 3-6 independent villages that owned cattle separately, and these formed 96 respondents who correspondingly had 96 cattle herds. A range of biologically plausible risk and deterministic factors of disease occurrence were studied and carefully examined their effect within the study populations and are initially expressed as proportions .Demographic characteristics of the respondents were as follows: Gender and female-headed households represented , while male-headed households represented  of the respondents. Similarly, 70% of the respondents were living within the buffer zones of the National Parks, with close to 60% coming from the eastern GMAs close to Lochinvar National park. The minimum number of people living in a household was 2, while the maximum number was 39.When considered independently, some of the significant factors that showed high proportional representativeness as contributing to disease transmission and maintenance included low Bacille Calmette–Guérin (BCG) vaccination at 29%,  the low level of knowledge on bTB at only 41%,  the high frequency of drinking unpasteurized milk at 81%, and  with the frequency of drinking at least 3 times a week, being a maintenance factor. Other plausible factors included low number of those boiling their milk at only 26%  and the high contact between domestic animals and wild animals at water points at 92%  .On univariate analysis of independent variables at herd level, four main factors were observed to have had an independent effect on TB herd status .Some variables had marginal effects while others had no effect. Of these variables, the most significant were contact with wildlife, being transhumant, moving cattle herds to the plains (thus moving of cattle to the plains for grazing for certain parts of the year), and cattle sharing grazing ground and drinking water as well as having animals being condemned at the abattoir, these came out very prominent.χ2=9.42, p=0.002). Moving animals to the plains were noted to be significantly associated with TB positivity . Similarly, cattle herds that were seen to share drinking water and share grazing grounds had a significant effect on finding a herd positive within it . Cattle herds from which animals taken to the abattoir for slaughter, had at least one animal condemned within the preceding 12 months before the study period, showed a significant effect on TB positivity   .χ2 (6)=5.2 and p=0.52. Cattle herds that were reported to be sharing grazing grounds and drinking water with wild animals concurrently had a significant effect on bTB status  given that contact with wildlife and having animals being condemned at the abattoir due to suspected TB are controlled , cattle sharing grazing ground, and drinking water as well as having animals being condemned at the abattoir due to suspected TB in the herd as factors having significant effect on herd bTB status . The Hosntrolled .Although there was no statistical difference between owners who had their cattle condemned at the abattoir due to suspected TB, or exhibiting TB lesions and those who had not , in general people who have had animals condemned at the abattoir tended to employ some precautionary measures or were known to be more responsive to bTB control programs because of the experience of losing out on having their animals condemned. Similarly, owners who had an experience of having their cattle condemned at the abattoir due to suspected TB, or exhibiting TB lesions, had a significant effect on bTB status given that contact with wildlife, being transhumant, and cattle sharing grazing ground and drinking water are controlled. Contact with wildlife as well as movement of cattle to the plains without actual sharing of drinking water and grazing pastures was found to have a protective effect on bTB herd status  and  given that the other two variables in the model are controlled.The results obtained from this study starting from trace back system of suspected abattoir bTB-positive cattle and their geospatial referencing have revealed the spatial distribution of the disease in cattle in Namwala District of Zambia. The results have particularly revealed that there is an ecological interplay in the spatial distribution of bTB in the high bTB prevalence area of Namwala District, which is itself a function of the existence of the reservoir hosts particularly in the eastern interface areas of the district. These results are further congruent with earlier traditional epidemiological results of bTB being more or less proportionately high in areas with Kafue Lechwe antelope populations .M. bovis has been characterized from Kafue Lechwe (Kobus Leche Kafuensis) [M. bovis is known to circulate in them. The prevalence is high in the interface area in Lochinvar National Park [It is worth noting that bTB in wildlife forms a stable reservoir such that fuensis) . The Kaffuensis) . Where anal Park . In factnal Park . Moreovenal Park  reportednal Park . FloodinA lot of research has previously been done implicating the Kafue Lechwe in the sustenance and spread of bTB. With regard to spread to livestock, mixing in the plains is the contributing factor . MoreoveDespite these findings and earlier studies, bTB remains a huge problem in Namwala District of Zambia, as was indicated from abattoir results. Across the study District of Namwala, the disease burden at herd level was found to be as high as 36.4% (95% CI 26.7-46.3%). From the gathered trace back data obtained from affected herds, there were factors which were identified as relevant clinical signs of bTB including emaciation and coughing in relation to at least one animal from the herd that had at least one animal condemned as having tuberculous lesions on slaughter at the two abattoirs in Namwala District. The individual prevalence based on animals condemned at the abattoirs in relation to total animals slaughtered was similarly high oscillating at 14% (95% CI=2.4-26.4%). While the disease has been persistently reported for some time now ,24,27,32It is well established that bTB has been known to affect humans for a long time. In fact, even to this day in spite of pasteurization and test and slaughter having been used successfully to bring down the incidence in the West, still, there are cases in countries such as the USA, especially in the binational communities . This deχ2 (6)=5.2, and p=0.52. Cattle herds that were reported to be sharing grazing grounds and drinking water with wild animals concurrently had a significant effect on bTB status  given that contact with wildlife and having animals being condemned at the abattoir due to suspected TB are controlled. While there were no tests done to ascertain the occurrence of bTB in humans during the study, it can be assumed that some human cases were related to the disease originating in animals. The assumption is borne out by the fact that at least 65% of those interviewed had been treated for TB and had bTB-positive herds. BTB has been known to be transmissible through drinking water to animals and in turn can be transmitted to humans if unpasteurized milk or contaminated carcasses are consumed. The contact with wildlife, which also leads to disease transmission to domestic animals, was as high as 52% and the sharing of drinking water at 91.67% in those interviewed. Contact with wildlife seems to be the primary factor since wildlife has been known to harbor bTB. Moreover, Lechwe antelopes found in Lochinvar National Park are important reservoirs of the disease. The influence of contact is highlighted by the maps generated in the study which indicates the geographical areas where the majority of bTB cases are originating from. From these maps, it is evident, given the larger circle, indicating the relative space covered by positive herds occurring along the GMAs of the Lochinvar National Park, the sanctuary of the Lechwe antelopes. The occurrence of bTB was more in the herds closer to Lochinvar National Park in the eastern region than in the western part of Namwala.In summary, when considered independently, some of the significant factors that showed high proportional representativeness as contributing to disease transmission and maintenance included the low level of knowledge on bTB at only 41%,  and the correspondingly high contact between domestic animals and wild animals at 92% . Other plausible factors for possible transmission to humans as a zoonotic risk included low number of those boiling their milk at only 26%  coupled with low level of BCG vaccination among children at 29% (95% CI 19.9-38.4%). The multiple logistic regression model identified contact with wildlife, being transhumant (thus moving of cattle to the plains for grazing for certain parts of the year), cattle sharing grazing ground and drinking water as well as having animals being condemned at the abattoir due to suspected TB in the herd as factors having significant effect on herd bTB status. The Hosmer-Lemeshow goodness-of-fit check showed that the model fitted the data well with n=96, Further data indicate that 70% of the respondents living within the buffer zones of the national parks indicated having lost an animal at the abattoir due to suspected tuberculous lesions. To test for association with the geographical area, a STATA command  for MoraWhile mapping is interested in finding patterns, no information was gathered on how synchronized bTB is in herds of Namwala as done in other studies . With thTransmission of bTB from wild animals to cattle, which poses a threat to humans has been widely studied . This stBased on the results obtained, we conclude that the spatial distribution of TB in Namwala follows the interface area where the Lechwe antelopes overlap with cattle in terms of grazing ground and watering points as captured. More importantly, the results showed that the prevalence is much higher in the eastern region of the district. The eastern region also happens to be closer to Lochinvar National Park, an important habitat of Lechwe antelopes which are known to harbor bTB. Spatial distribution of bTB is thus amenable to the use of GIS. Moreover, GIS can complement traditional epidemiological study methods such as questionnaire surveys.NFPT conceptualized the aim of the study, design, field study, georeferencing, and development of the first draft of the manuscript. BH helped in the conceptualization of the study, supervision, and revision of the manuscript. JBM helped in the conceptualization of the study, supervision, and revision of the manuscript. MM helped with field studies, supervision of data analysis, and revision of the manuscript. All the authors have read and approved the final manuscript.


Improvements in genetic and genomic technology have enabled field-deployable molecular laboratories and these have been deployed in a variety of epidemics that capture headlines. In this editorial, we highlight the importance of building physical and personnel capacity in low and middle income countries to deploy these technologies to improve diagnostics, understand transmission dynamics and provide feedback to endemic communities on actionable timelines. We describe our experiences with molecular field research on schistosomiasis, trypanosomiasis and rabies and urge the wider tropical medicine community to embrace these methods and help build capacity to benefit communities affected by endemic infectious diseases. Studying genetics no longer requires a laboratory facility with bright lights and hefty equipment: the technology and creative solutions facilitating PCR product-free and sterile environments nearly anywhere have enabled a rapid response to emerging pathogens. Although these epidemics dominate headlines, we argue that these opportunities should also be extended to improve diagnostics, understanding of transmission dynamics and to design more effective intervention strategies to target endemic infectious pathogens in low and middle income countries (LMICs). Moreover, building core capacity and routine molecular surveillance in the areas most burdened by infectious disease is critical to ensure epidemic preparedness.Ebola, yellow fever, Zika and COVID-19 outbreaks in the last few years have benefitted from real-time sequencing of pathogens to understand the transmission and evolution of these emerging viruses.There is immense potential to use new technologies and lowered equipment costs to move capacity into those regions that are most impacted by infectious diseases. Countless molecular work currently relies on shipping samples to complete processing, often with long delays and removed from those who collect samples and deal with pathogens on a daily basis. The declining cost of portable PCR machines, quantitative PCR and sequencers make them a more feasible option and several are robust to field conditions. These portable pieces of equipment mean that machines can stay in country and be moved on site to study specific endemic diseases in real time. They can even be used in stationary laboratories to build scalable molecular capacity at reference laboratories. Consumables are a real challenge, as we discuss below, but new protocols, tools and reagents are lowering hurdles for conducting molecular work.Trypanosoma brucei rhodesiense), we found that results obtained in the field with a modified extraction protocol and temperature-stable reagents were consistent with results later obtained with the same samples in a molecular lab at Makerere University  using modified solutions then used directly in PCR reactions  has developed accessible protocols to inactivate, amplify and sequence viral samples in the field in a robust, sensitive and safe manner. The development of portable laboratories has inspired many of the kit and processing pipelines described here. These researchers have used portable glove boxes to help contain infectious material before they are inactivated, and made their packing lists and operating protocols open for others to learn from. Portable glove boxes help maintain safety, keep the space sterile, and can be lightweight and packable  to a matter of days, providing rapid, actionable epidemiological insights for rabies control efforts.https://bomb.bio/about/).Despite these obvious benefits, we believe one of the biggest challenges in conducting molecular work is sourcing and paying for consumables. Local suppliers in LMICs offer a limited range of molecular reagents, items are often out of stock or take months to deliver and are prohibitively expensive. Importation regulations exacerbate the delays and expense of acquiring necessary reagents, and customs delays compromise the integrity of reagents. In our experience, airway bills, including duty tax and in-transit cold storage costs, often exceed the price of the reagents themselves. A secure, affordable supply chain will ultimately ensure the sustainability of molecular research in LMICs. While global companies continue to monopolise prices this will remain a major challenge to research implementation in these areas. Companies simply must do more to improve supply chains and the accessibility of their products. The wider research community is also beginning to develop low-cost ‘do-it-yourself’ alternatives for some reagents, which provide alternatives to some of these consumable needs (i.e. By bringing the lab to the sample, it is possible to avoid delays caused by shipping samples and long lag times in communicating results back to the affected communities. It is essential to build physical and human capital to carry out this work within endemic settings and ensure that consumables are not a limiting factor in conducting this work. Helicopter science limits the perspective of an endemic disease, and building confidence in the application of molecular methods throughout academic and government human and animal health institutions will only benefit from these advances.


Current pathology practice is being shaped by the increasing complexity of modern medicine, in particular of precision oncology, and major technological advances. In the “next-generation technologies era”, the pathologist has become the person responsible for the integration and interpretation of morphologic and molecular information and for the delivery of critical answers to diagnostic, prognostic and predictive queries, acquiring a prominent position in the molecular tumor boards. In 1953, Crick and Watson’s discovery of the DNA double-helix , Giovanni Battista Morgagni placed anatomo-clinical correlations at the heart of modern medicine [The performance of autopsy has been widely recognized for several decades as a fundamental part of routine pathology practice. In 1761, with the publication of medicine . The lesmedicine .However, since the last decades of the twentieth century, the rate of autopsies has decreased significantly, due to increasing confidence in ante-mortem diagnosis, more complex legislations regarding human tissues procedures and also insufficient priority given to autopsies by pathologists themselves, burdened with increasing workloads of surgical resections, biopsies, and cytology .The declining autopsy rate should be a source of concern among the scientific community, given its great potential for the advancement of medical knowledge and improvement of clinical practice. As demonstrated by the COVID-19 experience, the post-mortem examination, together with state-of-the-art molecular diagnostics, plays a central role in the diagnosis and clinical management of newly emerging diseases .Current pathology practice is being shaped by the increasing complexity of modern medicine and major technological advances . In the However, traditional organ, tissue and cellular pathology lays the foundation for the development of molecular pathology and, as demonstrated by the on-going COVID-19 pandemic, still provides essential tools to greatly enhance clinical practice.Current practicing and future pathologists are called on to actively incorporate molecular knowledge into their diagnostic armamentarium and deeply transform laboratory frameworks and pathology educational training programs.We are facing an epochal evolution of the figure of the pathologist who must necessarily find fertile ground both in laboratories, thanks to economic availability and mental openness towards innovation, and also in the training programs of young pathologists. Studies aimed at new technologies will be a fundamental background for the pathologist–in-training.Collaboration between different professional figures must acquire more and more importance in the context of the MTB. The first experiences of multidisciplinary teams in the molecular field are demonstrating how an integration between different knowledge in specialized fields such as clinical, molecular and surgical pathology and engineering can lead to an increasingly targeted therapy, to an increasingly personalized follow-up and a more precise cancer risk stratification.