Molecular Diagnosis Research Articles

A novel variant in the FLNB gene associated with spondylocarpotarsal synostosis syndrome.

Genetic disorders involved in skeleton system arise due to the disturbance in skeletal development, growth and homeostasis. Filamin B is an actin binding protein which is large dimeric protein which cross link actin cytoskeleton filaments into dynamic structure. A single nucleotide changes in the FLNB gene causes spondylocarpotarsal synostosis syndrome, a rare bone disorder due to which the fusion of carpels and tarsals synostosis occurred along with fused vertebrae. In the current study we investigated a family residing in north-western areas of Pakistan. The whole exome sequencing of proband was performed followed by Sanger sequencing of all family members of the subject to validate the variant segregation within the family. Bioinformatics tools were utilized to assess the pathogenicity of the variant. Whole Exome Sequencing revealed a novel variant (NM_001457: c.209C>T and p.Pro70Leu) in the FLNB gene which was homozygous missense mutation in the FLNB gene. The variant was further validated and visualized by Sanger sequencing and protein structure studies respectively as mentioned before. The findings have highlighted the importance of the molecular diagnosis in SCT (spondylocarpotarsal synostosis syndrome) for genetic risk counselling in consanguineous families.

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

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. 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. 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.

A prospective study on Rickettsia infections in Central India

Rickettsial infections are under-reported in India. The actual disease burden has been underestimated as non-specific serological tests are mostly used to diagnose these infections. The aim of this study was the detection of Rickettsia infections in clinical specimens by Real-time Polymerase Chain Reaction.A study was established to analyse blood samples taken from pyrexia of unknown origin (PUO) or undifferentiated fever cases. A total of 69 such patients, were referred/walked-in to our diagnostic centre, Indore, India between October 2021 and May 2023. The blood samples were collected in EDTA lavender (purple) top tube.Of the tested samples, 14 patients (20.3%) were positive for Rickettsia infection.Gender wise distribution revealed higher incidence (57%) of Rickettsial infections in male patients. Children were majorly infected (50% of cases); multiple infection was also noted in them, though severity was not high. Severe clinical manifestation including hospitalization was also noted in some patients. Few reports are available from India on molecular diagnosis of Rickettsia infection. Early-stage molecular diagnosis will help to stave off and prevent serious complications.

Molecular characterisation and diagnostics of the red ring nematode, Bursaphelenchus cocophilus, from Mexico

Molecular characterisation and diagnostics of the red ring nematode, Bursaphelenchus cocophilus, from Mexico

Molecular Diagnostics of Cryptococcus spp. and Immunomics of Cryptococcosis-Associated Immune Reconstitution Inflammatory Syndrome

Cryptococcal infection poses a significant global public health challenge, particularly in regions near the equator. In this review, we offer a succinct exploration of the Cryptococcus spp. genome and various molecular typing methods to assess the burden and genetic diversity of cryptococcal pathogens in the environment and clinical isolates. We delve into a detailed discussion on the molecular pathogenesis and diagnosis of immune reconstitution inflammatory syndrome (IRIS) associated with cryptococcosis, with a specific emphasis on cryptococcal meningitis IRIS (CM-IRIS). Our examination includes the recent literature on CM-IRIS, covering host cellulomics, proteomics, transcriptomics, and genomics.

Next step towards point-of-care molecular diagnosis of female genital schistosomiasis (FGS): evaluation of an instrument-free LAMP procedure

Detection of Schistosoma spp. DNA in gynaecological samples by quantitative real-time polymerase chain reaction (qPCR) is considered to be the reference diagnostic test for female genital schistosomiasis (FGS). However, qPCR needs expensive laboratory procedures and highly trained technicians. Loop-mediated amplification (LAMP) is a more field-friendly isothermal procedure for the detection of parasite-specific DNA, but it still requires electrically powered equipment. Here, we validated a Schistosoma haematobium-specific Sh-LAMP procedure and tested a fully instrument-free isothermal amplification using a novel low-cost, and reusable Temperature-cup (T-cup) device. Specific primers were selected based on published assays, targeting the ribosomal intergenic spacer (IGS) region of S. haematobium. Technical validation of the IGS-Sh-LAMP was performed using 20 negative controls, including DNA extracts of soil-transmitted helminths and S. mansoni, and a 10-fold dilution series (100–10−3) of DNA extracted from a single S. haematobium egg (n=4). For clinical validation, the IGS-Sh-LAMP was tested on 125 DNA samples extracted from vaginal swabs of a previous FGS study in Madagascar. Results were compared with the quantification cycle value (Cq) of the standard ITS-2 targeting qPCR. Single S. haematobium egg DNA up to a 10–2 dilution and an ITS-2 Cq <35 tested positive in the IGS-Sh-LAMP. The specificity was found to be excellent (100%). In the clinical samples, IGS-Sh-LAMP showed comparable results with the qPCR, with 35.2% and 33.6% positives, respectively, and a concordance of 79.2% (99/125). Of the 12 false-negatives, 5 corresponded to the 7 qPCR positive samples with very low DNA levels (Cq ≥35). On the other hand, IGS-Sh-LAMP detected 14 additional cases that were not detected by qPCR. The T-cup IGS-Sh-LAMP performance was evaluated in a representative sub-selection (n=10) of IGS-Sh-LAMP positive clinical samples. The T-cup IGS-Sh-LAMP was found to be a very user-friendly method, but in different runs, it missed 1 to 4 of the 10 IGS-Sh-LAMP positive samples, specifically those with a low DNA load. Our results show that the IGS-Sh-LAMP is a suitable alternative to the ITS-2 qPCR for the diagnosis of FGS in gynaecological samples, with high potential for the T-cup as a fully instrument-free isothermal amplification device for point-of-care diagnosis in low-resource settings.

Myxobolus acanthogobii Hoshina, 1952 and Myxobolus selari n. sp. (Myxosporea: Myxobolidae) infecting brain of commercial fishes in Terengganu, Malaysia.

Myxosporean infection in marine water fishes has drawn less attention than in freshwater fishes, which resulted in a higher taxonomic variety in freshwater in Malaysia. This study aimed to address the gap by conducting a myxosporean survey on two commercially significant marine fish species, Nemipterus furcosus (Valenciennes) (Eupercaria incertae sedis: Nemipteridae) and Selar crumenophthalmus (Bloch) (Carangiformes: Carangidae), collected from the northeastern part of peninsular Malaysia. During the examination of the organs, two distinct Myxobolus Bütschli, 1882 species were discovered in the brain tissue of these fishes, despite the absence of any observable pathological signs. The two Myxobolus species were characterized through morphometry, morphology, and analysis of partial small subunit ribosomal RNA (18S rDNA) gene. As a result, Myxobolus acanthogobii Hoshina, 1952, which infects 2.3% of N. furcosus, is synonymous with a myxobolid species commonly found in Japanese waters, based on its morphological traits, tissue tropism, and molecular diagnostics. Furthermore, a novel species, Myxobolus selari n. sp., was described, infecting the brain of one (11%) individual S. crumenophthalmus. This unique species displayed distinctive features, placing it within a well-supported subclade primarily comprising brain-infecting myxobolids. Maximum likelihood analysis further revealed the close relationships among these brain-infecting myxobolids, underscoring the significance of tissue tropism and host taxonomy for myxobolids. This study represents the initial documentation of Myxobolus species within the southern South China Sea, shedding light on the potential diversity of marine myxosporean in this region. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:7C400E35-7CB8-4DEE-92B7-F75FF3926441.

Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique

BackgroundMembers of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique.MethodsA total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses.ResultsAnaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters.ConclusionCattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

Molecular diagnostics of prostate cancer: impact of molecular tests.

Prostate cancer (PCa) is the second leading cause of cancer-related death among men. Prostate-specific antigen (PSA) testing is used in screening programs for early detection with a consequent reduction of PCa-specific mortality at the cost of overdiagnosis and overtreatment of the nonaggressive PCa. Recently, several assays have been commercially developed to implement PCa diagnosis, but they have not been included in both screening and diagnosis of PCa. This review aims to describe the actual and novel commercially available molecular biomarkers that can be used in PCa management to implement and tailor the screening and diagnosis of PCa.

Genomic medicine advances for brain tumors.

Cancer genome profiling has revealed important genetic alterations that serve as prognostic indicators and guides for treatment decisions, enabling precision medicine. The shift to molecular diagnosis of brain tumors, as reflected in the 2021 World Health Organization Classification of Tumors of the Central Nervous System, is a crucial role for treatment decision-making. This review discusses the significance and role of cancer genome profiling in precision medicine for malignant brain tumors, particularly gliomas. Furthermore, we explore the progress in cancer genome analysis, focusing on cancer gene panel testing, integration of genomic information in brain tumor classification, and hereditary tumors. Additionally, we discuss the transformative effect of genomic medicine on early detection, risk assessment, and precision medicine strategies. The tumor mutational burden in brain tumors is considered low, but the application of molecular targeted drugs, such as isocitrate dehydrogenase inhibitors, v-raf murine sarcoma viral oncogene homolog B1 inhibitors, fibroblast growth factor receptor inhibitors, neurotrophic tyrosine receptor kinase, mechanistic target of rapamycin inhibitors, and anti-programmed death receptor-1 antibody drugs are promising for glioma treatment. We also discuss the future prospects of molecular targeted drugs.

The functions and mechanisms of RNA modification in prostate: Current status and future perspectives

The increasing incidence and mortality of prostate cancer worldwide significantly impact the life span of male patients, emphasizing the urgency of understanding its pathogenic mechanism and associated molecular changes that regulate tumor progression for effective prevention and treatment. RNA modification, an important post-transcriptional regulatory process, profoundly influences tumor cell growth and metabolism, shaping cell fate. Over 170 RNA modification methods are known, with prominent research focusing on N6-methyladenosine, N7-methylguanosine, N1-methyladenosine, 5-methylcytidine, pseudouridine, and N4-acetylcytidine modifications. These alterations intricately regulate coding and non-coding RNA post-transcriptionally, affecting the stability of RNA and protein expression levels. This article delves into the latest advancements and challenges associated with various RNA modifications in prostate cancer tumor cells, tumor microenvironment, and core signaling molecule androgen receptors. It aims to provide new research targets and avenues for molecular diagnosis, treatment strategies, and improvement of the prognosis in prostate cancer.

The cost of genetic diagnosis of suspected hereditary pediatric cataracts with whole-exome sequencing from a middle-income country perspective: a mixed costing analysis.

Up to 25% of pediatric cataract cases are inherited. There is sparse information in the literature regarding the cost of whole-exome sequencing (WES) for suspected hereditary pediatric cataracts. Molecular diagnosis of suspected hereditary pediatric cataracts is important for comprehensive genetic counseling. We performed a partial economic evaluation with a mixed costing analysis, using reimbursement data and microcosting approach with a bottom-up technique to estimate the cost of using WES for genetic diagnosis of suspected hereditary pediatric cataracts from the perspective of the Brazilian governmental health care system. One hundred and ten participants from twenty-nine families in Rio de Janeiro (RJ) were included. Costs of consumables, staff and equipment were calculated. Two scenarios were created: (1) The reference scenario included patients from RJ with suspected hereditary pediatric cataracts plus two family members. (2) The alternative scenario considered other genetic diseases, resulting in 5,280 exams per month. Sensitivity analysis was also performed. In the reference scenario, the total cost per exam was 700.09 United States dollars (USD), and in the alternative scenario, the total cost was 559.23 USD. The cost of WES alone was 527.85 USD in the reference scenario and 386.98 USD in the alternative scenario. Sensitivity analysis revealed that the largest costs were associated with consumables in both scenarios. Economic evaluations can help inform policy decisions, especially in middle-income countries such as Brazil.

Development of a triplex quantitative reverse transcription-polymerase chain reaction for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus A

Porcine viral diarrhea is caused by many pathogens and can result in watery diarrhea, dehydration and death. Various detection methods, such as polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR), have been widely used for molecular diagnosis. We developed a triplex real-time quantitative reverse transcription PCR (qRT-PCR) for the simultaneous detection of three RNA viruses potentially associated with porcine viral diarrhea: porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus A (PoRVA). The triplex qRT-PCR had R2 values of 0.999 for the standard curves of PEDV, TGEV and PoRVA. Importantly, the limits of detection for PEDV, TGEV and PoRVA were 10 copies/μL. The specificity test showed that the triplex qRT-PCR detected these three pathogens specifically, without cross-reaction with other pathogens. In addition, the approach had good repeatability and reproducibility, with intra-and inter-assay coefficients of variation <1%. Finally, this approach was evaluated for its practicality in the field using 256 anal swab samples. The positive rates of PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. The co-infection rate of two or more pathogens was 2.73% (7/256). The new triplex qRT-PCR was compared with the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018) and showed 100% agreement for PEDV and TGEV and 95.70% for PoRVA. Therefore, the triplex qRT-PCR provided an accurate and sensitive method for identifying three potential RNA viruses for porcine viral diarrhea that could be applied to diagnosis, surveillance and epidemiological investigation.

Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19

Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

Pragmatic nationwide master observational trial based on genomic alterations in advanced solid tumors: KOrean Precision Medicine Networking Group Study of MOlecular profiling guided therapy based on genomic alterations in advanced Solid tumors (KOSMOS)-II study protocol KCSG AL-22–09

BackgroundNext-generation sequencing (NGS) has been introduced to many Korean institutions to support molecular diagnostics in cancer since 2017, when it became eligible for reimbursement by the National Health Insurance Service. However, the uptake of molecularly guided treatment (MGT) based on NGS results has been limited because of stringent regulations regarding prescriptions outside of approved indications, a lack of clinical trial opportunities, and limited access to molecular tumor boards (MTB) at most institutions. The KOSMOS-II study was designed to demonstrate the feasibility and effectiveness of MGT, informed by MTBs, using a nationwide precision medicine platform.MethodsThe KOSMOS-II trial is a large-scale nationwide master observational study. It involves a framework for screening patients with metastatic solid tumors for actionable genetic alterations based on local NGS testing. It recommends MGT through a remote and centralized MTB meeting held biweekly. MGT can include one of the following options: Tier 1, the therapeutic use of investigational drugs targeting genetic alterations such as ALK, EGFR, ERBB2, BRAF, FH, ROS1, and RET, or those with high tumor mutational burden; Tier 2, comprising drugs with approved indications or those permitted for treatment outside of the indications approved by the Health Insurance Review and Assessment Service of Korea; Tier 3, involving clinical trials matching the genetic alterations recommended by the MTB. Given the anticipated proportion of patients receiving MGT in the range of 50% ± 3.25%, this study aims to enroll 1,000 patients. Patients must have progressed to one or more lines of therapy and undergone NGS before enrollment.DiscussionThis pragmatic master protocol provides a mass-screening platform for rare genetic alterations and high-quality real-world data. Collateral clinical trials, translational studies, and clinico-genomic databases will contribute to generating evidence for drug repositioning and the development of new biomarkers.Trial registrationNCT05525858.

Hemizygous deletion of CDKN2A/B with p16 immuno-negative and methylthioadenosine phosphorylase retention predicts poor prognosis in IDH-mutant adult glioma

Abstract Background Homozygous deletion of the tumor suppression genes cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) is a strong adverse prognostic factor in IDH-mutant gliomas, particularly astrocytoma. However, the impact of hemizygous deletion of CDKN2A/B is unknown. Furthermore, the influence of CDKN2A/B status in IDH-mutant and 1p/19q-codeleted oligodendroglioma remains controversial. We examined the impact of CDKN2A/B status classification, including hemizygous deletions, on the prognosis of IDH-mutant gliomas. Methods We enrolled 101 adults with IDH-mutant glioma between December 2002 and November 2021. CDKN2A/B deletion was evaluated with multiplex ligation-dependent probe amplification (MLPA). Immunohistochemical analysis of p16/MTAP and promoter methylation analysis with methylation-specific MLPA was performed for cases with CDKN2A/B deletion. Kaplan−Meier plots and Cox proportion hazards model analyses were performed to evaluate the impact on overall (OS) and progression-free survival. Results Of 101 cases, 12 and 4 were classified as hemizygous and homozygous deletion, respectively. Immunohistochemistry revealed p16-negative and MTAP retention in cases with hemizygous deletion, whereas homozygous deletions had p16-negative and MTAP loss. In astrocytoma, OS was shorter in the order of homozygous deletion, hemizygous deletion, and copy-neutral groups (median OS: 38.5, 59.5, and 93.1 months, respectively). Multivariate analysis revealed hazard ratios of 9.30 (p=0.0191) and 2.44 (p=0.0943) for homozygous and hemizygous deletions, respectively. Conclusions CDKN2A/B hemizygous deletions exerted a negative impact on OS in astrocytoma. Immunohistochemistry of p16/MTAP can be utilized to validate hemizygous or homozygous deletions in combination with conventional molecular diagnosis.

A REVIEW OF NANOPARTICLE INNOVATIONS IN CANCER THERAPY: IMPLICATIONS, TARGETING MECHANISMS AND CLINICAL PROSPECTS

The main reason for morbidity and death globally is cancer, which has a complex pathophysiology. There are several traditional treatments for cancer, including chemotherapy, radiation therapy, targeted therapies, and immunotherapies. Multiple drug resistance, cytotoxicity, and lack of specificity pose significant challenges to cancer treatments. Molecular diagnostics and cancer treatment have been transformed by nanotechnology. For cancer treatment, nanoparticles (1–100 nm) are ideal because they are biocompatible, have low toxicity, excellent stability, high permeability, are precise and stable, and can deliver clear and accurate results. There are several main categories of nanoparticles. When it comes to the delivery of nanoparticle drugs, tumour characteristics and the tumour environment are considered. As well as providing advantages over conventional cancer treatments, nanoparticles prevent multidrug resistance, further overcoming their limitations. As new mechanisms are unravelled in studying multidrug resistance, nanoparticles are becoming more critical. Nano formulations have gained a new perspective on cancer treatment due to their many therapeutic applications. The number of approved nanodrugs has not increased significantly despite most research being conducted in vivo and in vitro. A review of nanoparticle oncological implications, targeting mechanisms, and approved nanotherapeutics is presented here. A current perspective on clinical translation is also provided, highlighting its advantages and challenges.

First molecular detection of Borrelia sp. in tapirs (Tapirus terrestris).

Borrelia theileri is a tick-borne spirochete causative agent of fever, apathy and reduced food consumption in cattle. Molecular diagnosis has expanded the understanding of Borrelia theileri with new hosts and geographical locations being described. The present study aimed to describe the first molecular detection of B. theileri in wild tapirs (Tapirus terrestris) from South America. Blood DNA samples obtained from 99 tapirs sampled in Pantanal (n = 61) and Cerrado (n = 38) biomes were screened using a qPCR assay based on the 16S rRNA gene of Borrelia sp. Positive samples in the qPCR assay were subjected to PCR assays to allow characterization of fragments from 16S rRNA and flaB genes. Two (2/99; 2.0%) animals from Pantanal biome were positive in the qPCR and one sample presented bands of expected size for the flaB protocol. Amplicons from this sample were successfully cloned and sequenced. In the phylogenetic analysis, Borrelia sp. from T. terrestris grouped together with B. theileri sequences previously detected in Rhipicephalus microplus ticks and cattle from Minas Gerais State in Brazil, Rhipicephalus geigyi from Mali, and R. microplus and Haemaphysalis sulcata from Pakistan. This finding contributes to our knowledge regarding susceptible hosts species for B. theileri. More studies are necessary to understand the potential effects of B. theileri on tapir's health.

Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Deoxyguanosine kinase deficiency: natural history and liver transplant outcome

Abstract Autosomal recessive pathogenetic variants in the DGUOK gene cause deficiency of deoxyguanosine kinase activity and mitochondrial deoxynucleotides pool imbalance, consequently, leading to quantitative and/or qualitative impairment of mitochondrial DNA synthesis. Typically, patients present early-onset liver failure with or without neurological involvement and a clinical course rapidly progressing to death. This is an international multicentre study aiming to provide a retrospective natural history of deoxyguanosine kinase deficient patients. A systematic literature review from January 2001 to June 2023 was conducted. Physicians of research centers or clinicians all around the world caring for previously reported patients were contacted to provide follow-up information or additional clinical, biochemical, histological/histochemical, and molecular genetics data for unreported cases with a confirmed molecular diagnosis of deoxyguanosine kinase deficiency. A cohort of 202 genetically confirmed patients, 36 unreported, and 166 from a systematic literature review, were analyzed. Patients had a neonatal onset (≤ 1 month) in 55.7% of cases, infantile (>1 month and ≤ 1 year) in 32.3%, pediatric (>1 year and ≤18 years) in 2.5%, and adult (>18 years) in 9.5%. Kaplan-Meier analysis showed statistically different survival rates (p < 0.0001) among the four age groups with the highest mortality for neonatal onset. Based on the clinical phenotype, we defined four different clinical subtypes: hepatocerebral (58.8%), isolated hepatopathy (21.9%), hepatomyoencephalopathy (9.6%), and isolated myopathy (9.6%). Muscle involvement was predominant in adult-onset cases whereas liver dysfunction causes morbidity and mortality in early-onset patients with a median survival of less than 1 year. No genotype-phenotype correlation was identified. Liver transplant significantly modified the survival rate in 26 treated patients when compared with untreated. Only six patients had additional mild neurological signs after liver transplant. In conclusion, deoxyguanosine kinase deficiency is a disease spectrum with a prevalent liver and brain tissue-specificity in neonatal and infantile-onset patients and muscle tissue-specificity in adult-onset cases. Our study provides clinical, molecular genetics, and biochemical data for early diagnosis, clinical trial planning, and immediate intervention with liver transplant and/or nucleoside supplementation.