French National Reference Laboratory Research Articles

Performances of the BD MAX™ CDIFF assay for the detection of toxigenic Clostridioides difficile using Cary-Blair preserved samples

Toxigenic Clostridioides difficile infection (CDI) is a high concern because of its increasing prevalence, among both nosocomial infection and community-acquired infection contexts. A total of 1320 prospective stool samples collected on FecalSwab™ (Cary-Blair medium) were screened using BD Max™ CDIFF assay (BDM). Results were compared to the routine algorithm including immunochromatographic testing for glutamate dehydrogenase (GDH) and toxin A/B (tox) using C. DIFF QUIK CHEK COMPLETE® followed by Xpert C. difficile assay in case of GDH+/tox- results. The prevalence of toxigenic C. difficile was estimated to 5.3% (n=70/1320) using the routine algorithm but to 7.4% (n = 98/1320) using BDM. The overall agreement between BDM and the routine algorithm was 97.4%. After resolution of discrepant results between BDM and the algorithm using Xpert C. difficile assay (unless already perform) and toxigenic culture performed by the French National Reference Laboratory, the overall agreement was increased to 99.4%, demonstrating the excellent performances of the test.

Positive predictive value of seroconversion or positive rubella IgM in diagnosis of maternal rubella infection: Seven-years review of French National Reference Laboratory for Rubella.

In France, as in most developed countries, childbearing age women are routinely screened for rubella antibodies to identify and vaccinate susceptible women. Immunity to rubella is usually determined by measuring the rubella virus-specific immunoglobulin G (RV-IgG). In case of seroconversion for RV-IgG and/or positive RVIgM during pregnancy, laboratories usually send serum samples to the French National Reference Laboratory (FNRL) for Rubella in order to perform complementary investigations and confirm or exclude rubella infection during pregnancy. Our aim is to report results of these investigations during a seven-year period (2013-2019) and evaluate the positive predictive value (PPV) of RV-IgG seroconversion or positive RV-IgM to diagnose maternal rubella infection in France. Between 2013 and 2019, 5398 serum samples collected from 4104 pregnant women, were addressed to FNRL because of RV-IgG seroconversion (N=899) or positive RV-IgM (N=3205). Additional serological tests were performed, mainly immunoblot (to look for the presence of anti-E1 protective antibody) and RV-IgG avidity (to exclude or confirm primary infection). Overall, for 3724/4104 (90.8 %) women, rubella primary-infection during pregnancy was formally excluded and maternal rubella primary-infection was only confirmed in 46/4104 (1.1 %) cases. Clinicians and biologists should be particularly aware that RV-IgG seroconversion or positive RV-IgM, in the current context of low RV circulation in France are most often not rubella primary infections. PPV of seroconversion to assess maternal rubella primary infection is as low as 0.2 % (95 % CI: 0 %; 0.5 %) and PPV of positive RV-IgM is only of 1.4 % (95 % CI: 0.99 %; 1.81 %).

Identification of Neisseria meningitidis by MALDI-TOF MS may not be reliable

ObjectivesThe matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique is increasingly used in hospital laboratories for routine identification of microorganisms. However, its performance is variable, particularly for highly variable species such as Neisseria meningitidis. Reliable identification of N. meningitidis is crucial for the management of invasive meningococcal disease by rapid implementation of treatment and preventive measures among close contacts. We assessed and improved N. meningitidis identification by MALDI-TOF MS by enriching the databases with reference strains identified using whole genome sequencing (WGS) as a reference standard. MethodsWe first built a collection of 24 strains from several species of the Neisseria genus that we characterized by WGS. This collection was added to the available database to test by MALDI-TOF MS another collection of 32 clinical isolates received between 2015 and 2017 at the French National Reference Laboratory for Meningococci. ResultsUsing the commercially available library of mass spectrometry profiles, only 67% (95% confidence interval (CI), 47–82) concordance was observed at the species level between MALDI-TOF MS and WGS characterization. However, when the new enriched reference collection was used on the second subset of isolates, the identification of N. meningitidis was significantly improved (p 0.0016), showing 92% (95% CI, 75–98) specificity while that of the manufacturer's database alone was 52% (95% CI, 34–70). ConclusionsOur data highlight the need to update the available MALDI-TOF MS database with high-quality references to enhance the identification of N. meningitidis and avoid unwarranted preventive measures or missing identification.

Use of FTA® card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa

Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA® card technology. The DNA samples were taken by applying the FTA® paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA® samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing.

ABSTRACTListeria monocytogenes is a ubiquitous bacterium that may cause the foodborne illness listeriosis. Only a small amount of data about the population genetic structure of strains isolated from food is available. This study aimed to provide an accurate view of the L. monocytogenes food strain population in France. From 1999 to 2014, 1,894 L. monocytogenes strains were isolated from food at the French National Reference Laboratory for L. monocytogenes and classified according to the five risk food matrices defined by the European Food Safety Authority (EFSA). A total of 396 strains were selected on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France. The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i) there was not a clear association between ST9 and ST121 and the food matrices, (ii) serotype IIc, ST8, and ST4 were associated with meat products, and (iii) ST13 was associated with dairy products. Of the two major STs, ST121 was the ST that included the fewest clinical strains, which might indicate lower virulence. This observation may be directly relevant for refining risk analysis models for the better management of food safety.IMPORTANCE This study showed a very useful backward compatibility between PFGE and MLST for surveillance. The results enabled better understanding of the population structure of L. monocytogenes strains isolated from food and management of the health risks associated with L. monocytogenes food strains. Moreover, this work provided an accurate view of L. monocytogenes strain populations associated with specific food matrices. We clearly showed that some STs were associated with food matrices, such as meat, meat products, and dairy products. We opened the way to source attribution modeling in order to quantify the relative importance of the main food matrices.

Susceptibility of enterohaemorrhagic Escherichia coli to azithromycin in France and analysis of resistance mechanisms.

Antibiotic therapy is not recommended during enterohaemorrhagic Escherichia coli (EHEC) infection. However, the potential benefit of azithromycin has recently been described. This study was conducted to evaluate the macrolide susceptibility of EHEC isolates from France and to analyse the mechanisms of resistance to azithromycin in EHEC. Strains from patients with EHEC infections were collected by the associated French National Reference Laboratory for E. coli from 2004 to 2014. Antimicrobial susceptibility testing was performed by disc diffusion and Etest methods. For strains presenting macrolide resistance, antibiotic resistance genes were searched for by PCR. Genetic transfer was performed by conjugation and plasmid analysis was done by Southern-blot hybridization after PFGE. We tested 508 isolates of EHEC. Azithromycin MICs ranged between 0.25 and 16 mg/L (median = 3 mg/L), except for two atypical strains, 34396 and 36493, for which MICs were >256 mg/L. Plasmid transferability of macrolide resistance was demonstrated. Strain 34396, of serotype O106:H18, harboured two macrolide resistance genes [mph(A) and erm(B)] and two other antimicrobial resistance genes (blaDHA-1 for β-lactam resistance and qnrB4 for quinolone resistance). mph(A), blaDHA-1 and qnrB4 were localized on the same plasmid. Strain 36493 belonging to the highly virulent O26:H11 EHEC clonal group harboured a plasmid containing mph(A). For the first time we describe plasmid-borne macrolide resistance genes in EHEC strains. Dissemination of such plasmids may threaten the potential benefit of azithromycin during the management of patients infected or colonized with EHEC. Determination of azithromycin MICs should be considered prior to its therapeutic use and 16 mg/L may be used as the epidemiological cut-off value.

Benefits of PCR and decentralization of diagnosis in regional laboratories in the management of Bluetongue in France.

Since 1998, Bluetongue virus (BTV) serotypes 1, 2, 4, 6, 8, 9, 11 and 16 have spread throughout Europe. In 2006, BTV serotype 8 (BTV‑8) emerged unexpectedly in Northern Europe, in countries such as Belgium, France, Germany, Luxembourg, and the Netherlands, to spread rapidly in the following year throughout the rest of Europe. In 2007, BTV‑1 spread in Southern Europe, in Spain and in South of France. In 2008, 2 more BTV serotypes were detected in Northern Europe: BTV‑6 in the Netherlands and in Germany, and BTV‑11 in Belgium. The European incursion of BTV has caused considerable economic losses, including direct losses from mortality and reduced production, as well as indirect losses generated by ensuing bans on trade of ruminants between infected and non-infected areas. Given the significance of the disease, all affected countries have established control and eradication measures that have evolved together with the availability of detection and prevention tools such as Polymerase Chain Reaction (PCR) tests and vaccines, respectively. This paper describes how the French National Reference Laboratory for BT has managed diagnosis during the fast and massive spread of BTV‑1 and 8 in 2007 and 2008.

Two clusters of ciguatera fish poisoning in Paris, France, related to tropical fish imported from the French Caribbean by travelers.

Ciguatera fish poisoning (CFP) is a food-borne illness due to the consumption of reef fish containing pathogenic toxins. CFP is endemic to tropical areas and may be described in travelers in non-endemic areas. We describe two clusters of autochthonous cases of CFP in Paris, France. They were related to two fish caught in Guadeloupe (French West Indies) and consumed in Paris after being air-transported in a cooler. In both cases, fish flesh was analyzed and the presence of ciguatoxins by mouse bioassay (MBA) was confirmed. The first cluster involved eight individuals among whom five presented gastrointestinal symptoms and four presented neurological symptoms after consuming barracuda flesh (Sphyraena barracuda). The second cluster involved a couple who consumed a grey snapper (Lutjanus griseus). Most of them consulted at different emergency departments in the region of Paris. CFP may be seen in non-traveler patients outside endemic countries resulting from imported species of fish. Thus, CFP may be undiagnosed as physicians are not aware of this tropical disease outside endemic countries. The detection of ciguatoxins by MBA in the French National Reference Laboratory is useful in the confirmation of the diagnosis.

Clues for a standardised thermal-optical protocol for the assessment of organic and elemental carbon within ambient air particulate matter

Abstract. Along with some research networking programmes, the European Directive 2008/50/CE requires chemical speciation of fine aerosol (PM2.5), including elemental (EC) and organic carbon (OC), at a few rural sites in European countries. Meanwhile, the thermal-optical technique is considered by the European and US networking agencies and normalisation bodies as a reference method to quantify EC–OC collected on filters. Although commonly used for many years, this technique still suffers from a lack of information on the comparability of the different analytical protocols (temperature protocols, type of optical correction) currently applied in the laboratories. To better evaluate the EC–OC data set quality and related uncertainties, the French National Reference Laboratory for Ambient Air Quality Monitoring (LCSQA) organised an EC–OC comparison exercise for French laboratories using different thermal-optical methods (five laboratories only). While there is good agreement on total carbon (TC) measurements among all participants, some differences can be observed on the EC / TC ratio, even among laboratories using the same thermal protocol. These results led to further tests on the influence of the optical correction: results obtained from different European laboratories confirmed that there were higher differences between OCTOT and OCTOR measured with NIOSH 5040 in comparison to EUSAAR-2. Also, striking differences between ECTOT / ECTOR ratios can be observed when comparing results obtained for rural and urban samples, with ECTOT being 50% lower than ECTOR at rural sites whereas it is only 20% lower at urban sites. The PM chemical composition could explain these differences but the way it influences the EC–OC measurement is not clear and needs further investigation. Meanwhile, some additional tests seem to indicate an influence of oven soiling on the EC–OC measurement data quality. This highlights the necessity to follow the laser signal decrease with time and its impact on measurements. Nevertheless, this should be confirmed by further experiments, involving more samples and various instruments, to enable statistical processing. All these results provide insights to determine the quality of EC–OC analytical methods and may contribute to the work toward establishing method standardisation.

Genotyping of French Bacillus anthracis strains based on 31-loci multi locus VNTR analysis: epidemiology, marker evaluation, and update of the internet genotype database.

Background Bacillus anthracis is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described.ResultsMLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site.ConclusionsThe present report provides a wide coverage of the genetic diversity of naturally occurring B. anthracis strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as B. anthracis.

The organization of proficiency testing in plant pathology (qualitative methods of analysis) according to the ISO/IEC 17043: example of the French national reference laboratory

The use of methods of analysis capable of producing reliable analytical results is a prerequisite to the effective control of quarantine plant pathogens. Proficiency testing is considered to be one of the most reliable ways to verify and coordinate analytical results. As a French national reference laboratory in plant pathology, the Anses Plant Health Laboratory organizes proficiency tests in order to ensure that officially approved laboratories (certified by government services) are capable of producing reliable analytical results for the detection of plant pathogens. Proficiency tests in plant pathology have a number of notable features including the processing of qualitative results. This paper presents the experience of the Anses Plant Health Laboratory’s Unit for Tropical Pests and Diseases (LSV-RAPT) as an organizer of proficiency tests in plant pathology. The LSV-RAPT has gained recognition for the methodology it has developed in the form of accreditation as a proficiency testing provider according to the ISO/IEC 17043. The methodology can be applied to many other disciplines that use qualitative detection methods.

Five years’ experience of classical swine fever polymerase chain reaction ring trials in France

Since 2004, the French National Reference Laboratory for classical swine fever (CSF) has conducted an annual proficiency test (PT) to evaluate the ability of local veterinary laboratories to perform real-time polymerase chain reaction (PCR) for CSF virus. The results of five years of testing (2004-2008) are described here. The PT was conducted under blind conditions on 20 samples. The same batch of samples was used for all five years. The number of laboratories that analysed the samples increased from four in 2004 to 13 in 2008. The results of the PT showed the following: cross-contamination between samples and deficiencies in RNA preparation can occur even in experienced laboratories; sample homogeneity should be checked carefully before selection; samples stored at-80 degrees C for several years remain stable; and poor shipment conditions do not damage the samples with regard to detection of CSF virus genome. These results will enable redesign of the panel to improve the overall quality of the PT, which will encourage laboratories to check and improve their PCR procedures and expertise. This is an excellent way to determine laboratory performance.

Strayed dogs sentinels ofTrichinella britoviinfection in Kosovo

Strayed dogs sentinels of<i>Trichinella britovi</i>infection in Kosovo

Investigation on HPV DNA detection and genotyping practices used in French laboratories in 2009

The French National reference Laboratory for Human papillomavirus (HPV) performed in 2009 a national study in order to review the methods used to detect and identify HR HPV genotypes in microbiology laboratories. Results from this study show a great diversity in volumes of samples treated in laboratories. Among clinical indications, the most frequent is a result of ASC-US at a Pap smear. This indication in the only one covered by the National Public Insurance System and is mostly performed in laboratories from private sector. Other indications mainly correspond to research programs and are performed in public Hospitals. This study allowed also to review the adequacy between the liquid based cytology samples and the assays used for direct detection of HR HPV or identification of the genotypes present in the sample. The right tests were not carried in the right solution storage according to the recommendations from different HPV testing assays. National recommendations should be elaborated in order to improve the performance of the test used.

Contamination levels for lead, cadmium and mercury in marine gastropods, echinoderms and tunicates

As part of a specific monitoring programme, total lead (Pb), cadmium (Cd) and mercury (Hg) concentrations in marine gastropods (common winkle, common whelk, abalone and murex), echinoderms (purple sea urchin and black sea cucumber) and tunicates (an ascidian species) were investigated by the French National Reference Laboratory (NRL) for trace elements in foodstuffs of animal origin. These elements were analysed by atomic absorption spectrometry in order to evaluate the safety of these species for human consumption. The highest levels of Hg and Cd were found in murexes, 0.185 mg kg −1 and 0.853 mg kg −1, respectively; whereas the highest level of Pb was detected in ascidians (0.505 mg kg −1). Hg and Pb concentrations were always below the maximum levels set by the European legislation (0.5 mg Hg kg −1 and 1.5 mg Pb kg −1 wet mass) and for Cd, only 2 samples of murex (2.09 ± 0.42 mg kg −1 and 2.33 ± 0.46 mg kg −1) exceeded the maximum level set by French legislation of 2.0 mg kg −1 wet mass.

Le diagnostic rétrospectif de l’infection congénitale à cytomégalovirus sur le sang séché des cartes de Guthrie : l’expérience en France

Systematic screening for cytomegalovirus congenital infection is not performed in France. For children with hearing loss or other neurological CMV compatible symptoms, retrospective diagnosis is possible by PCR detection of CMV DNA in dried blood spot of neonatal Guthrie cards. We report here the results obtained with this technique in the French national reference laboratory for cytomegalovirus.

Evaluation of 10-years French NRL proficiency tests for lead, cadmium and mercury analysis in foodstuff of animal origin

The French National Reference Laboratory (NRL) for heavy metals in foodstuffs of animal origin at the French Food Safety Agency (AFSSA) regularly organises proficiency tests schemes (PTS) focused on the quantification of the three elements lead (Pb), cadmium (Cd) and mercury (Hg), for the 23 departmental laboratories of the network of laboratories, under its responsibility. The NRL prepares test materials to promote comparability of data and support harmonisation of official analytical procedures in the case of routine analysis. When target values for the analytes are set, the PTS also provide an objective assessment and improvement of the analytical trueness in the daily work of the laboratories. The long-term performance of the network was examined using results of 10 years of PTS in various matrices from different foodstuffs of animal origin (fish, meat and milk products), with 3 different official methods, using Flame- and Electro-Thermal-AAS techniques. In all cases, participants were requested to carry out 3 independent measurements of Pb, Cd and Hg. Moreover, the assessment of the PTS was based on the Z-score approach. From 1998 to 2008, this evaluation applied to 690 results obtained in 41 PTS indicated a frequency distribution of the Z-score value for the three elements Pb, Cd and Hg of 89.2%, 94.5% and 97.7% of acceptable results, respectively. The results of this balance sheet indicated the homogeneity and ability of the laboratories network to provide comparable results of similar quality. Comparison with results obtained by a primary method were presented as complementary information to determine the interest to organize, in the future, PTS based on a certified value.

Les phénotypes érythrocytaires rares : un enjeu de santé publique

A rare blood group is usually defined as the absence of a high prevalence antigen or the absence of several antigens within a single blood group system, if its prevalence in France is 4/1000 or less in the general population. An individual with a rare blood phenotype can develop a naturally-occurring or immune antibody corresponding to his rare specificity. In case an extremely low stock of compatible blood is available at the national level, a so-called “transfusion deadlock” is described. Most of the individuals with a rare blood group are coincidently identified when a routine pretransfusion testing or pregnancy follow-up is performed, if the antibody(ies) corresponding to the rare specificity is(are) present. Other individuals are discovered following a systematic red cell typing, or family investigations in siblings. One hundred and twenty-one rare blood specificities and 42 rare blood genotypes are currently defined at the French National Reference Laboratory for Blood Groups (CNRGS–Paris). The French national registry of individuals with a rare blood phenotype/genotype includes about 9600 people, who are urged to regularly donate blood for the National Rare Blood Bank. This bank, based on a homologous blood transfusion program, is in charge of the long-term storage of rare frozen blood units, that can only be delivered after receiving authorization from the CNRGS. The global and individual care management of the individuals with a rare blood group, concerning potentially several hundred thousand people in France, requires a close cooperation between all the protagonists within the transfusion chain.

Laboratory based hepatitis A surveillance in New Caledonia: from an endemic to an epidemic pattern (1986-2007)

This study aimed at describing the evolution of the epidemiological pattern of hepatitis A in New Caledonia since 1986 and the recent epidemic which occurred in 2005-2006, regarding particularly its demographic and virological aspects and the public health response implemented. The annual or monthly activity records for Hepatitis A sero-diagnostic performed at the Pasteur Institute of New Caledonia were processed in a retrospective analysis (9723 samples tested for the detection of IgM to hepatitis A). Over the 2004-2006 period, a phylogenetic study of representative strains from New Caledonia and other Pacific islands was carried out by the French National Reference Laboratory for hepatitis A (Paul-Brousse hospital, Villejuif, France). The continuous improvement of hygiene that occurred in New Caledonia during the last two decades led to a dramatic drop in the frequency of hepatitis A among patients tested, ranging from an average value of 79 cases (14%) for the 1986-1999 period to 0 case from 2002. However, in 2005, a strong increasing number of confirmed cases was notified, mainly among young people (78% were under the age of 20). In 2006, this epidemic reached the island of Futuna where it involved more than 1% of the total population (56 cases). The phylogenetic study has confirmed the clonality of the virus circulating during this epidemic, not related to other regional strains (Fiji, Vanuatu, New Zealand) nor with a New Caledonian strain from the previous endemic period. This transition situation, with persistence of a high epidemic risk, should encourage the health authorities to implement adapted response strategies, based in particular on systematic case declaration and targeted immunisation programmes.

Late Occuring Severe Autoimmune Hemolytic Anemia in a Liver Transplanted Child.

INTRODUCTION Autoimmune haemolytic anaemia (AIHA) can complicate solid organ as well as bone marrow transplantation both in children and adults. We describe the first case report of a child with AIHA due to mixed type warm-acting IgM and warm IgG auto-antibodies, 8 months after liver transplantation.CASE REPORT A sixteen-month old boy (A Rh D positive blood group,), 8 months after an orthotopic liver transplant (full cadaveric liver, male A Rh D negative) presented with fever and moderate hepatitis. Two weeks after this episode he developed severe anemia with a Hb level of 39 g/L. His red cells were A1 Rh(D) positive. Plasma testing could not be interpreted due to positive reactions with all test cells. Screening for irregular antibodies was positive. The direct antiglobulin test was repeatedly negative. Further analysis revealed a cold agglutinin with low titre (8 at 15°C) but high thermal amplitude (22–37°C) although of IgM nature, and the absence of any underlying alloantibodies. Only Rh null red blood cells were not agglutinated by the autoantibody. Therapy included keeping body temperature over 37° C, transfusions of A Rh(D) positive warmed crossmatched blood units, intravenous immunoglobulins (1g/kg/d x 5 days), and methylprednisolone (20 mg/kg/d). Tacrolimus was replaced by cyclosporin A. Further investigations revealed panagglutinating IgG autoantibodies in the plasma and on the erythrocytes and the monospecific direct antiglobulin test became positive for IgM, IgG, C3c and C3d. French National Reference Laboratory for Immuno-Hematology and Rare Blood Groups confirmed these findings and showed both the IgM and IgG autoantibodies to be directed against the Rh proteins (anti-RH29 antibody) and to be strongly active at 37°C. Crossmatched group O Rh(D) positive red blood cell (RBC) units were transfused, however with limited effect and progressive haemolysis. Hb level dropped to 33 g/L. One cryopreserved O Rh null RBC unit was obtained from the French National Rare Blood Bank. Rituximab® (375 mg/m2 once weekly) (anti-CD 20 monoclonal antibody) was also introduced. Immediately following this transfusion and 24 hours after the first dose of rituximab, Hb levels increased and were stable at 80 g/L. No further transfusions were needed and the haemolytic parameters normalized slowly. Total 4 doses of rituximab were administered.Ten weeks after admission, the child could be discharged. At 18 months' follow-up, there is no recurrence of AIHA.DISCUSSION Only few cases of AIHA due to warm acting “cold” agglutinins have been described, generally resulting in death. AIHA in patients with liver transplant has been previously reported, mainly due to warm autoantibodies or to classical cold agglutinin disease associated with viral infections, lymphoproliferative disease or autoimmune disorders. Our case is interesting in that this unusual AIHA occurred not only in a liver transplanted child, but also late after transplantation. Although extensive investigations revealed no aetiology, the hypothesis of a viral infection-triggered AIHA with first an IgM, then a mixture of IgM and IgG autoantibodies directed against the same epitope is plausible. This case illustrates the efficacy of rituximab in AIHA not responding to first-line therapy and the importance of international collaboration to provide extremely rare compatible blood units.