Automatic patient-level recognition of four Plasmodium species on thin blood smear by a real-time detection transformer (RT-DETR) object detection algorithm: a proof-of-concept and evaluation.

Malaria remains a global health problem, with 247 million cases and 619,000 deaths in 2021. Diagnosis of Plasmodium species is important for administering the appropriate treatment. The gold-standard diagnosis for accurate species identification remains the thin blood smear. Nevertheless, this method is time-consuming and requires highly skilled and trained microscopists. To overcome these issues, new diagnostic tools based on deep learning are emerging. This study aimed to evaluate the performances of a real-time detection transformer (RT-DETR) object detection algorithm to discriminate Plasmodium species on thin blood smear images. The algorithm was trained and validated on a data set consisting in 24,720 images from 475 thin blood smears corresponding to 2,002,597 labels. Performances were calculated with a test data set of 4,508 images from 170 smears corresponding to 358,825 labels coming from six French university hospitals. At the patient level, the RT-DETR algorithm exhibited an overall accuracy of 79.4% (135/170) with a recall of 74% (40/54) and 81.9% (95/116) for negative and positive smears, respectively. Among Plasmodium-positive smears, the global accuracy was 82.7% (91/110) with a recall of 90% (38/42), 81.8% (18/22), and 76.1% (35/46) for P. falciparum, P. malariae, and P. ovale/vivax, respectively. The RT-DETR model achieved a World Health Organization (WHO) competence level 2 for species identification. Besides, the RT-DETR algorithm may be run in real-time on low-cost devices such as a smartphone and could be suitable for deployment in low-resource setting areas lacking microscopy experts.IMPORTANCEMalaria remains a global health problem, with 247 million cases and 619,000 deaths in 2021. Diagnosis of Plasmodium species is important for administering the appropriate treatment. The gold-standard diagnosis for accurate species identification remains the thin blood smear. Nevertheless, this method is time-consuming and requires highly skilled and trained microscopists. To overcome these issues, new diagnostic tools based on deep learning are emerging. This study aimed to evaluate the performances of a real-time detection transformer (RT-DETR) object detection algorithm to discriminate Plasmodium species on thin blood smear images. Performances were calculated with a test data set of 4,508 images from 170 smears coming from six French university hospitals. The RT-DETR model achieved a World Health Organization (WHO) competence level 2 for species identification. Besides, the RT-DETR algorithm may be run in real-time on low-cost devices and could be suitable for deployment in low-resource setting areas.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine-pyrimethamine in central Africa: a cross-sectional study.

BACKGROUND: Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. METHODS: Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. FINDINGS: Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. INTERPRETATION: Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. FUNDING: Toulouse Institute for Infectious and Inflammatory Diseases.

Evaluation of the Performance of the Novodiag® Stool Parasites Assay for the Detection of Intestinal Protozoa and Microsporidia.

OBJECTIVES: We aimed to assess the performance of the Novodiag® Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections. METHODS: A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia. RESULTS: Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for Blastocystis hominis, Entamoeba histolytica and Cyclospora cayetanensis but was lower for Giardia intestinalis (85.2%) and ≤50% for Cystoisospora belli and Dientamoeba fragilis. In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for G. intestinalis, D. fragilis, Cryptosporidium spp. and E. histolytica. Sensitivity was 100% for Enterocytozoon bieneusi, but none of the five samples containing Encephalitozoon spp. were detected. CONCLUSIONS: The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR.

Effect of sulfadoxine-pyrimethamine chemoprophylaxis in pregnant women on selection of the new P. falciparum dhps quintuple mutant carrying the I431V mutation.

BACKGROUND: A new mutation in the Plasmodium falciparum dihydropteroate synthetase gene (pfdhps), I431V, has been identified in several countries of Central and West Africa. This mutation is mostly found in association with four other SNPs on pfdhps (S436A, A437G, A581G and A613S), forming a quintuple mutant (vagKgs) and almost always associated with the Plasmodium falciparum dihydrofolate reductase gene (pfdhfr) CirnI (C50R, N51I, S108N) triple mutant. To date, nothing is known about the impact of this new pfdhps genotype on sulfadoxine-pyrimethamine (SP) resistance. OBJECTIVES: We sought to assess the prevalence of this pfdhps vagKgs quintuple mutant in two groups of pregnant women with malaria, one that took intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) and one that did not. METHODS: The pfdhfr and pfdhps genes from Plasmodium falciparum isolates collected in Yaoundé (Cameroon) from pregnant women with symptomatic malaria under IPTp-SP or not, were sequenced. RESULTS: Of 159 patients evaluated, 70 had already taken SP during pregnancy and 89 had never taken SP. Only the vagKgs allele was significantly overrepresented in the SP+ group (21.4% versus 3.4%; P < 0.001), whereas the ISgKAA mutant, widely distributed in this area and known to be less susceptible to SP, tended to be less abundant in this group (48.6% versus 64.0%; P = 0.0503). CONCLUSIONS: We found a strong overrepresentation of the CirnI/vagKgs haplotype in the IPTp-SP pregnant group, suggesting a high level of resistance of this mutant to SP. This could compromise not only the effectiveness of IPTp-SP but also the seasonal malaria chemoprevention of young children, now widely implemented.

Fatal mucormycosis and aspergillosis coinfection associated with haemophagocytic lymphohistiocytosis: A case report and literature review.

Invasive mould infections are life-threatening and mainly occur in immunocompromised patients. Whereas aspergillosis is described during haemophagocytic lymphohistiocytosis (HLH), only a few cases of concomitant mucormycosis with HLH have been reported. Here, we present an uncommon coinfection of mucormycosis and aspergillosis associated with HLH probably due to a varicella zoster virus (VZV) viraemia which was unresponsive to triple antifungal therapy (liposomal amphotericin B combined with isavuconazole and caspofungin). A review of the cases of mucormycosis with HLH showed that this uncommon association was always lethal and underscored the relevance of screening for mould infections in patients with HLH.

Diagnosis of Congenital Toxoplasmosis: Performance of Four IgG and IgM Automated Assays at Birth in a Tricentric Evaluation.

For postnatal diagnosis of congenital toxoplasmosis (CT), the gold standard for the detection of anti-Toxoplasma IgM in newborns relies on the immunosorbent agglutination assay (ISAGA), which is manufactured from whole Toxoplasma parasites that become difficult to maintain. For IgG, only the Platelia assay provides a validated assay for cord blood according to the manufacturer, allowing its use in this context. We compared the analytical performance of four commercialized automated assays, Platelia, Abbott, Vidas, and Liaison, for the detection of IgG and IgM in the cord blood or peripheral blood of newborns from women infected during pregnancy. The assays were performed on samples from 509 newborns, collected from the university hospitals of Montpellier, Nîmes, and Toulouse. For IgM, the four assays appeared to be sufficiently informative to be used for congenital toxoplasmosis diagnosis (area under the curve [AUC] > 0.8, receiver operating characteristic [ROC] analysis), with Platelia showing the best performance, similar to ISAGA with regard to accuracy (83%). For the Vidas (76%), Abbott (75%), and Liaison (74%) assays, the accuracy was significantly lower. Maternal treatment significantly decreased the sensitivity of all the assays. For IgG, the four evaluated assays showed a sensitivity of over 90%, with Abbott (95%) and Liaison (94%), exhibiting a significantly higher sensitivity than Platelia (90%). Furthermore, Abbott showed its superiority in the cases of maternal infection during the third trimester. In the context of the newborns of mothers infected by Toxoplasma gondii during pregnancy, to ensure efficient care, Platelia and Abbott seemed to be the most suitable reference tests for the detection of IgM for the former and IgG for the latter.

A Clustered Case Series of Mucorales Detection in Respiratory Samples from COVID-19 Patients in Intensive Care, France, August to September 2021.

While COVID-19-associated pulmonary aspergillosis is now well described in developed countries, COVID-19-associated mucormycosis (CAM) has seemed to remain quite rare in Europe. A retrospective study was performed between March 2020 to September 2021 among COVID-19 adult patients in the intensive care unit (ICU) at Toulouse Hospital (Southern France). PCR screening on respiratory samples, which target Aspergillus or Mucorales DNA, were performed, and the number of fungal detections was evaluated monthly during the study period. During the 19 months of the study, 44 (20.3%) COVID-19 ICU patients had a positive PCR for Aspergillus, an overall rate in keeping with the incidence of ICU COVID-19 patients. Ten patients (7.1%) had a positive Mucorales PCR over the same period. Surprisingly, 9/10 had a positive Mucor/Rhizopus PCR in August-September 2021, during the fourth Delta SARS-CoV-2 variant wave. Epidemic investigations have identified a probable environmental cause linked to construction works in the vicinity of the ICU (high levels of airborne spores due to the mistaken interruption of preventive humidification and summer temperature). Even if CAM are apparently rare in Europe, a cluster can also develop in industrialised countries when environmental conditions (especially during construction work) are associated with a high number of COVID-19 patients in the ICU.

New Insights into Blood Circulating Lymphocytes in Human Pneumocystis Pneumonia.

The host lymphocyte response is decisive in Pneumocystis pneumonia (PCP) pathophysiology but little is known of the specific roles of lymphocyte subpopulations in this fungal infection. Peripheral NK, NKT, B, TCD4+ and TCD8+ subpopulations were compared by immunophenotyping between 20 patients diagnosed with PCP (PCP(+)] and 20 uninfected immunosuppressed patients (PCP(-)). Among PCP(+) subjects, the lymphocyte populations were also compared between surviving and deceased patients. Low B cell count (<40 cells/µL) was more frequent in PCP(+) than in PCP(-) patients (p = 0.03), while there was no difference for the TCD4 count. Among the PCP(+) group, the 7 deceased patients had lower Th1 (p = 0.02) and Tc1 (p = 0.03) populations, higher Th2 response (p = 0.03), higher effector TCD8 (p < 0.01), lower central memory TCD8 (p = 0.04) and reduced NK cells (p = 0.02) compared with the 13 survivors. Th1/Th2 ratio < 17, CD8 Tc1 < 44%, effector TCD8 < 25%, central memory TCD8 < 4%, NK cells < 50 cells/µL and total lymphocytes < 0.75 G/L were associated with a higher risk of mortality (p = 0.003, p = 0.007, p = 0.0007, p = 0.004, p = 0.02 and p = 0.019, respectively). The traditional analysis of TCD4 and TCD8 populations may be insufficient in the context of PCP. It could be completed by using B cells to predict the risk of PCP, and by using lymphocyte subpopulations or total lymphocyte count, which are easy to obtain in all health care facilities, to evaluate PCP prognosis.

Real-time PCR for diagnosis of imported schistosomiasis.

BACKGROUND: The diagnosis of schistosomiasis currently relies on microscopic detection of schistosome eggs in stool or urine samples and serological assays. The poor sensitivity of standard microscopic procedures performed in routine laboratories, makes molecular detection methods of increasing interest. The aim of the study was to evaluate two in-house real-time Schistosoma PCRs, targeting respectively S. mansoni [Sm] and S. haematobium [Sh] in excreta, biopsies and sera as potential tools to diagnose active infections and to monitor treatment efficacy. METHODS: Schistosoma PCRs were performed on 412 samples (124 urine, 86 stools, 8 biopsies, 194 sera) from patients with suspected schistosomiasis, before anti-parasitic treatment. Results were compared to microscopic examination and serological assays (enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination (HA) and Western Blot (WB) assay). RESULTS: Compared to microscopy, PCRs significantly increased the sensitivity of diagnosis, from 4% to 10.5% and from 33.7% to 48.8%, for Sh in urine and Sm in stools, respectively. The overall sensitivity of PCR on serum samples was 72.7% and reached 94.1% in patients with positive excreta (microscopy). The specificity of serum PCR was 98.9%. After treatment, serum PCR positivity rates slowly declined from 93.8% at day 30 to 8.3% at day 360, whereas antibody detection remained positive after 1 year. CONCLUSION: Schistosoma PCRs clearly outperform standard microscopy on stools and urine and could be part of reference methods combined with WB-based serology, which remains a gold standard for initial diagnosis. When serological assays are positive and microscopy is negative, serum PCRs provide species information to guide further clinical exploration. Biomarkers such as DNA and antibodies are of limited relevance for early treatment monitoring but serum PCR could be useful when performed at least 1 year after treatment to help confirm a cured infection.

Síndrome serotoninérgico con rabdomiolisis y convulsiones asociado a la interacción farmacológica entre venlafaxina y amoxicilinaclavulánico / Serotonin syndrome, rhabdomyolysis and convulsion associated with drug interaction between venlafaxine and amoxicillin/clavulanic acid

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