Clinical evaluation of the Roche Elecsys CMV IgG Avidity assay.

Congenital cytomegalovirus (CMV) infection has potentially severe consequences in newborns. The testing of pregnant women for CMV-specific antibodies may be useful for the identification of women at risk of transmitting the infection to the fetus. The determination of CMV IgG avidity helps to establish the timing of infection as IgG avidity matures during the course of infection. This study examines the performance of the Elecsys CMV IgG Avidity assay using preselected samples from patients at different phases of CMV infection. The Elecsys CMV IgG Avidity assay was tested at three sites using sequential samples from patients with recent primary CMV infection, as well as single samples from patients with recent primary or past CMV infection. The Elecsys assay discriminated well between early (low avidity) and late (high avidity) phases of infection in sequential serum samples. Overall, 98.8% of low-avidity samples corresponded to infection onset <180 days before sampling and 77.8% of all high-avidity results corresponded to infection onset >90 days before sampling. The assay's sensitivity was 90-97%, with specificity ranging from 89 to 100%, depending on the consideration of gray-zone avidity values. Single samples from recent primary or past infection showed similar distributions of avidity results. The Elecsys CMV IgG Avidity assay results are in agreement with preselected samples from patients with primary or past CMV infection, showing that the test is an adequate predictor of the phase of infection.

Analytical issues possibly affecting the performance of commercial human cytomegalovirus IgG avidity assays.

In the majority of cytomegalovirus (CMV) immunoglobulin G (IgG) avidity assays, avidity determination can be performed on the entire CMV IgG measurable positive concentration range. However, in some exceptional samples with very low IgG levels, inappropriately low avidity indexes have been described. In this study, we addressed some possible causes and the clinical importance of these inappropriately low avidity indexes. We compared VIDAS (bioMérieux), Liaison (DiaSorin), and Architect (Abbott) CMV IgG avidity assays on 129 samples from patients with past CMV infections, focusing on samples with low IgG levels. Inappropriately low avidity samples were further evaluated using seven different urea-based IgG avidity assays. We confirmed that inappropriately low avidity indexes in samples with very low IgG levels occur, but are rare. We could show that this phenomenon is not confined to a single assay and that assays employing chaotropic agents are affected more frequently and profoundly. In situations where the CMV IgG avidity is performed on CMV immunoglobulin M (IgM)-negative samples, the avidity index should be interpreted cautiously in cases of very low CMV IgG levels, whatever the technique used.

A series of 238 cytomegalovirus primary infections during pregnancy: description and outcome.

OBJECTIVE: To analyze the outcome of maternal primary cytomegalovirus (CMV) infection. METHODS: Retrospective analysis of a cohort of 238 patients with maternal primary CMV infection detected at routine screening. The cases were managed with serial ultrasound (US) scans, and amniocentesis was performed in 36.1% of cases. All prenatal results were confirmed at birth. RESULTS: The average age was 31.9 (18-44) years. Patients were symptomatic in 21% of cases. The rate of intrauterine transmission was 24.9%, and it was 8.8%, 19%, 30.6%, 34.1% and 40% in the preconceptional period, the periconceptional period, and the first, second and third trimesters of pregnancy, respectively (p = 0.025). There was a significantly higher risk of US abnormalities when maternal infection occurred during the preconceptional or periconceptional period and the first trimester compared with later (p < 0.001). Because of US abnormalities, pregnancy was terminated in 18 cases at the parents' request. Three infected newborns were symptomatic; all three cases were suspected at US before birth. We did not observe any symptomatic fetal infection when maternal infection occurred after 14 weeks of gestation. A number of clinically asymptomatic cases (5.5%) developed hearing loss. CONCLUSION: The rate of materno fetal transmission is linearly correlated to the gestational age at infection. No severe case of congenital infection was observed if maternal infection occurred after 14 weeks of gestation.

Clinical evaluation of new automated cytomegalovirus IgM and IgG assays for the Elecsys(®) analyser platform.

Cytomegalovirus (CMV) is a leading cause of physical and neurological abnormalities in newborns. Hence, the diagnosis of CMV infection in pregnant women is necessary in order to allow appropriate management of their pregnancy. New assays have been developed for the Roche Elecsys® immunoassay platform that detect CMV-specific immunoglobulin (Ig)M and IgG, with the IgM assay designed to target IgM produced at the start of infection rather than IgM persisting later in infection. This study aimed to evaluate the performance of the new assays compared with other commercial kits widely distributed in laboratories. The performance of the Elecsys and comparator CMV IgM and IgG assays was assessed using 967 preselected patient samples characterised by CMV infection status, as well as being compared using 1,668 unselected clinical samples. The Elecsys CMV IgM and IgG assays performed consistently with comparator assays using the preselected samples. The Elecsys CMV IgM assay showed improved sensitivity compared with the Enzygnost® assay in primary infection (91.2 % vs. 79.4 %) and improved specificity over the Architect® assay in potentially cross-reacting samples (94.1 % vs. 82.4 %). The Elecsys IgM assay reported fewer positive results in the later stages of CMV infection compared with ETI-CYTOK-M ELISA, while the Elecsys IgG assay reported slightly fewer negative results in the early stages of infection compared with ETI-CYTOK-G ELISA. There was good agreement between Elecsys and comparator assays using unselected clinical samples (range 90.4-99.4 %). The Elecsys CMV IgM and IgG assays compare well with routinely used assays and are suitable for clinical use.

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.

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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). RESULTS: 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. CONCLUSION: 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 %).

Aluminum and vaccines: Current state of knowledge.

French people have never been so wary about vaccines. The use of aluminum salts in vaccine adjuvants to enhance effectiveness is one of the major reasons for this lack of confidence. The direct toxicity of aluminum is often put forward. Direct toxicity of aluminum has long been known-especially with occupational exposure-to be associated with characteristic clinical manifestations and increased blood aluminum level. Intoxication related to the excessive amount of an element in the body, whether be it lead poisoning following exposure to lead or mercury poisoning for instance, is always associated with metal increase in biological media. To date no link has been established between the direct toxicity of aluminum and vaccines. Aluminum levels in biological media of vaccinated subjects are not different from those of unvaccinated subjects. This is consistent with the very small amount of aluminum contained in one dose of vaccine. Indirect toxicity of aluminum was suggested to explain macrophagic myofasciitis in humans in 2011, a disease that could be mediated by an autoimmune/autoinflammatory mechanism. This hypothesis has recently been refuted in a large pharmaco-epidemiological study proving that aluminum-containing adjuvants of vaccines are not responsible for this autoimmune/autoinflammatory syndrome.

Evolution of awareness and knowledge of congenital cytomegalovirus infection among health care providers in France between 2011 and 2018.

BACKGROUND: Cytomegalovirus (CMV) is the most frequent cause of congenital viral infection. Approximately 1 % of newborns are congenitally infected and in up to 10 % of them the consequences are severe. Antenatal and postnatal treatments, although promising, are still under evaluation. Hygiene counseling to prevent CMV infection is important and should be systematic. OBJECTIVE: To evaluate health care providers' awareness of CMV maternal and congenital infection in France. STUDY DESIGN: A questionnaire on CMV infection was sent in 2018 by e-mail to obstetricians, pediatricians, midwives and laboratory physicians, and members of medical or midwifery associations. We evaluated their knowledge concerning CMV epidemiology, transmission, symptoms in adults, newborns and long-term effects (scores from 0 to 30) and compared the results with those of our 2012 published study. RESULTS: Of the 597 respondents who completed the questionnaire, 91 % were unaware of the precise transmission route of CMV, 33 % wrongly thought thatin utero therapy for congenital CMV infection was a current standard of care in France, and less than half were familiar with the HAS (Haute Autorité de Santé) and CNGOF (Collège National des Gynécologues et Obstétriciens Français) recommendations. When respondents' knowledge of CMV was greater, patients were given more hygiene counseling. Between 2011 and 2018, knowledge improved among doctors and midwives concerning the route of transmission, the symptoms in adults, and the long-term effects of CMV infection. CONCLUSIONS: Knowledge is improving among healthcare providers, but gaps remain. To bridge these gaps, health care providers should improve their knowledge about congenital CMV by various means: medical reviews, continuing medical education, meetings, conferences, the Internet. Moreover, greater knowledge will allow for more effective counseling of pregnant women, as recommended by HCSP and CNGOF in France.

A 2-year study on cytomegalovirus infection during pregnancy in a French hospital.

OBJECTIVES: To evaluate the proportion of pregnant women agreeing to cytomegalovirus (CMV) serologic screening. To collect data on CMV infection during pregnancy. DESIGN: Prospective study. SETTING: During two years, all pregnant women were informed on CMV infection. If the patient agreed, serological testing was performed around 12 weeks of gestation (WG) and, if negative, redone around 36 WG. POPULATION: Four thousand two hundred and eighty-seven pregnant women followed from 12 weeks to delivery. METHODS: If the first CMV serologic test was negative, detailed hygiene information was given to the parents. Diagnosis of primary infection was based on the detection of CMV-G, CMV-M and low CMV-G avidity index. When maternal infection was confirmed, diagnosis of CMV congenital infection was done in the newborns by urine culture within the three days following birth. Crude infection-rate data consisted of the number of CMV infection cases and person-time units for both exposed to hygiene CMV information (12 to 36 WG) and unexposed pregnant women (first 12 WG). MAIN OUTCOME MEASURES: Rate of CMV seropositive and seronegative women. Rate of women agreeing for screening. Rate of primary infection. Rate of seroconversion. Number of CMV-infected newborns. RESULTS: Among the 4287 women followed, 3792 were either seronegative or with an unknown immune status. 96.7% out of them agreed for screening. 53.2% were initially CMV-specific IgG negative. Primary infection was detected in nine women between 0 and 12 WG (0.46%) and seroconversion was diagnosed in five women between 12 and 36 WG (0.26%) (mid P = 0.02, 95% CI [1.07-13.6]). CONCLUSIONS: If clear information on CMV infection during pregnancy is given, patients frequently agree to screening. The rate of seroconversion after information, observed in this study, is low after counselling.

[Cytomegalovirus and pregnancy]. / Cytomégalovirus et grossesse.

Cytomegalovirus infection is the most common viral infection transmitted from the mother to the fetus. Congenital CMV infection occurs in approximately 1% of all newborns. The most serious fetal damages mainly occur (10%) after primary infection during pregnancy. Clinically apparent infections are characterized by involvement of multiple organs particularly the central nervous system with severe sequelae such as mental retardation, deafness and ocular defects. Diagnosis of CMV maternal/fetal infection could be justified by its frequency and its potential severity but the absence of reliable prognostic markers of congenital CMV disease makes difficult its management during pregnancy. A better knowledge of the physiopathology of CMV placental infection, correct counselling, identification of prognostic markers of fetal CMV infection may avoid the occurrence of the most severe fetal infections before development of safe and efficient vaccines and treatments.

Determination of rubella virus-specific humoral and cell-mediated immunity in pregnant women with negative or equivocal rubella-specific IgG in routine screening.

BACKGROUND: Immunity to rubella-virus (RV) is commonly determined by measuring specific IgG (RV-IgG). However, RV-IgG results may be different and even discordant, depending on the assay used. Cell-mediated immunity is not routinely investigated for diagnostic purposes. OBJECTIVES: Our aim was to investigate humoral and cellular immunity of women with negative or equivocal RV-IgG before, and after post-partum vaccination. STUDY DESIGN: A total of 186 pregnant women were included in the study. During pregnancy, humoral immunity was investigated with two RV-IgG immunoassays, an immunoblot and a T-cell mediated immunity test. In the post-partum vaccination period, measuring RV-IgM and RV-IgG avidity allowed us to determine whether women raised a primary or a secondary immune response. RESULTS: Before vaccination, 52.2% women, supposed to be susceptible, had positive anti-E1 RV-IgG indicating strong evidence of previous exposure to RV. All (100%) pregant women who had a positive immunoblot before immunization raised a secondary immune response to vaccination, and 96.8% who had a negative immunoblot before immunization, raised a primary immune response to vaccination. All women who raised a primary immune response after vaccination had negative anti-E1 RV-IgG and negative cell-mediated immunity. DISCUSSION: These results indicate that individuals can have evidence of protective immunity against rubella despite negative RV-IgG.

Cytomegalovirus non-primary infection during pregnancy. Can serology help with diagnosis?

Diagnosis of cytomegalovirus (CMV) primary infection is reliable, but diagnosis of CMV non-primary infection (NPI) is questionable. Our aim is to highlight the difficulties met in diagnosis of CMV NPI. We illustrate that in proven cases of CMV NPI, very different serologic and molecular patterns may be observed and that routine serologic testing may fail to help with diagnosis. These results point out that many data available in literature concerning the prevalence of NPI, materno-fetal transmission rates and consequences of NPI may be wrong. We need to know how frequently they occur, are transmitted and cause fetal damages. Diagnosis of NPI must be improved, along with our understanding of the mechanisms leading to intrauterine CMV transmission and congenital infection in babies born to women with preexisting immunity.

Early control of HIV replication in primary HIV-1 infection treated with antiretroviral drugs and pegylated IFN alpha: results from the Primoferon A (ANRS 086) Study.

IFN alpha has both antiviral and immunostimulating properties. The ANRS086 Primoferon A Study evaluated in 12 patients with primary HIV infection the tolerance and efficacy of an early and transient administration of pegylated IFN alpha, in addition to highly active antiretroviral therapy. Tolerance was good, and this regimen allowed the early control of HIV replication and rapid decay of the viral reservoir. These results support the initiation of comparative studies with pegylated INF alpha in primary HIV infection.

An indirect immunofluorescence staining procedure for detection of human Fc gamma receptors on streptococci.

Fc gamma receptors on streptococci are usually revealed by hemagglutinating techniques (IgG coated red blood cells) or uptake of radiolabeled IgG. The results obtained with these methods are not always satisfactory. For this reason, we developed a technique involving indirect immunofluorescence staining. Bacterial smears were treated with human Fc gamma fragment and their binding to streptococcal Fc gamma receptors was revealed by a fluorescent F(ab')2 fragment of anti-human Fc gamma sheep antibodies purified on an IgG immunosorbent. These purified sheep F(ab')2 fragments did not contain any IgG nor Fc gamma as shown by SDS polyacrylamide gel electrophoresis. Under these conditions indirect immunofluorescence staining was a highly specific and sensitive method of detecting Fc gamma receptors on streptococci. Distribution of Fc gamma receptors was studied in 237 streptococcal strains of human origin belonging to groups A, B, C, D and G; these receptors were also looked for in 21 strains of alpha-hemolytic streptococci which did not possess the group carbohydrate and 12 strains of pneumococci. Fc gamma receptors were found only in group A, C and G streptococci, but all strains of these groups did not possess Fc gamma receptors.

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies.

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM antimicrobial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of nonabsorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.

Enterovirus in sudden unexpected deaths in infants.

BACKGROUND: Conventional approaches to virus detection failed to provide convincing evidence of a viral etiology in sudden unexplained deaths in infants (SUDI). Many viruses may not have been detected by the routinely used methods; among them enteroviruses (EV) have seldom been found in SUDI. METHODS: In this study EV were sought directly in stools, in pharyngeal and tracheal samples and in myocardial and lung tissues, by using a nested PCR; they were also sought indirectly by detecting IgM antibodies with a new capture immunoassay. Twenty-four SUDI cases were divided into two groups: Group I, certainly associated with; or Group II, not associated with clinical, biologic or histologic signs of viral infection. RESULTS: EV were found in stools but their prevalence was not significantly different between Group I and Group II (20 and 22.2%, respectively). On the contrary EV were detected in respiratory tract and/or lung samples in 53.8% of infants of Group I and in none of Group II. Anti-EV IgM antibodies were detected in 55.5% of infants of Group I and in none of Group II. CONCLUSIONS: These results indicate that EV infection may be specifically associated with the subgroup of SUDI with viral signs, raising the question of its role in this condition.

Diagnosis and prognostic markers of HCMV infection.

Human cytomegalovirus (HCMV) infections are frequently observed as well in immunocompetent as in immunocompromised patients. Diagnostic techniques for HCMV detection have greatly improved during the recent years. Detection of HCMV by culture has been bettered by centrifugating samples in a shell vial and by using monoclonal antibody to the immediate early antigen. Detection of antigens in leucocytes was facilitated by using whole blood instead of leucocytes separated by dextran sedimentation. Detection of HCMV recently sharpened by using molecular biology methods mainly based on the detection of the genome. Under certain circumstances, and especially in pregnant women, diagnosis of HCMV infection is essentially based on the detection of IgG and IgM antibodies. However, as IgM antibody is not a marker of primary infection, complementary tests are needed to help in the datation of the infection. Among them, the measurement of IgG avidity greatly improved the diagnosis of primary infections. In immunocompromised patients, very sensitive techniques are needed to diagnose HCMV infections. Detection of antigenemia and HCMV DNA are the methods of choice for an early detection of the infection. Diagnosis of HCMV-organ diseases largely depends on the infected organ and the presence of HCMV in the organ is, sometimes, difficult to interpret. Today, diagnosis of congenital infections is possible by detecting HCMV in the amniotic fluid. However, in order to reliably detect HCMV, amniocentesis must be performed after 21 week's gestation and at least 6 weeks after seroconversion. As all HCMV infections do not induce disease, prognostic markers are needed, as well in immunocompromised patients as in fetuses. Many studies were conducted in immunocompromised patients to find prognostic markers of HCMV infection in order to introduce preemptive therapy when needed. It seems that quantitative determination of HCMV DNA could be very useful to predict HCMV disease. Qualitative determination of mRNA could also be useful as a prognostic marker of HCMV disease. Determination of viral genotypes is more controversial. As for the infected fetuses, little was published about the prediction of sequelae. Some factors, such as viral load in amniotic fluid or in fetal blood or level of IgM antibody in fetal blood, may be predictive of sequelae, but these data require further studies.

Congenital HCMV infection: a collaborative and comparative study of virus detection in amniotic fluid by culture and by PCR.

Cytomegalovirus (HCMV) infection is the leading cause of congenital virus infection in developed countries, affecting an estimated 1% of births. This antenatal infection can cause serious sequelae. Strategies for prevention and treatment must, therefore, be agreed upon, entailing a preliminary performance assessment of antenatal virus diagnosis techniques. Between 1992 and 1999, HCMV serology status was established for 19456 pregnant women in four French hospitals. Seronegative patients (55.4%) were given serology screening, and antenatal diagnosis was given to 152 women who had shown seroconversion during their pregnancies (1.4%). The detection of HCMV transmission from mother to fetus was finally established in 95 cases, using polymerase chain reaction (PCR) and viral culture methods for detecting HCMV in the amniotic fluid. These results were compared with viral culture of children's urine after birth, enabling us to distinguish between children really infected in utero (30%) and non-infected children (70%). The results of the virus culture and those of PCR were identical in 94 of the 95 cases, with one discrepancy (culture-/PCR+). The two diagnosis techniques had identical sensitivity (72%), with culture proving slightly more specific than PCR (98.4% as opposed to 96.9%). Positive prediction values for culture and for PCR were, respectively, 95.6 and 91.3%. Antenatal virus diagnosis on amniotic fluid was negative with both techniques in 8 out of 29 cases of children born with HCMV infection (VPN=89%). Over half of these wrongly negative results can be explained by amniocentesis carried out too early in the pregnancy or too early with respect to the mother's primary infection.

Specificity and sensitivity of the IgM capture immunoassay: studies of possible factors inducing false positive or false negative results.

The specificity and sensitivity of the IgM-capture immunoassay (IgM-CI) were evaluated for detection of rubella specific IgM and hepatitis B core (HBc) specific IgM. For rubella specific IgM, antibodies bound to the solid phase were detected by haemadsorption and for HBc specific IgM, by using HBc antigen (HBcAg) and radiolabelled IgG anti-HBc. Rheumatoid factor (RF) was found to interfere in the test for HBc specific IgM because IgM-RF bound to the solid phase reacted with aggregated radiolabelled HBc specific IgG. This false positive reaction did not occur when radiolabelled F(ab')2 was used instead of the whole IgG molecule. HBcAg purified from biological fluids might be coated with host IgG and under these conditions, HBcAg could react with RF. It was also demonstrated that high levels of IgG antibodies could interfere with IgG anti-mu coated-surface by means of non-immunological protein-protein interactions. In fact, IgG did not interfere in the rubella assay, whereas it did in the very sensitive anti-HBc test. To prevent this false-positive reaction, different dilution media were tested. Only the addition of non-specific IgG and fetal calf serum (FCS), to the dilution medium, seems to improve the specificity of the test. Furthermore, in order to decrease this non-specific IgG-IgG interaction and an occasional prozoning phenomenon, the dilution of serum to be tested was taken into account. Parameters considered to decrease sensitivity were also studied. RF, anti-F(ab')2 antibodies and non-specific IgM did not decrease significantly the sensitivity of the assay.

Microbiology in sudden infant death syndrome (SIDS) and other childhood deaths.

The usefulness of post-mortem microbiology in the assessment of sudden unexpected deaths in infants and children has been debated by many pathologists. In our centre, microbiological investigations have been part of the post-mortem protocol for investigation of sudden deaths in infants and children for the past 12 years. The objective of this study was to review the microbiological findings for infants and children examined by our unit during the past 4 years in relation to gross and histological findings of the autopsy and the medical and social histories of the children. We reviewed 57 consecutive sudden deaths in infants and children examined by our Referral Centre between November 1994 and October 1998. These 57 sudden deaths were aged from 1 day to 4 years and 9 months including 40 cases of sudden infant death syndrome (SIDS) and 17 non-SIDS deaths. Results of the microbiological investigations of tissues and body fluids were assessed during the case review with reference to histological shock signs, severe gastric aspiration, and signs of acute thymic involution. Bacteria alone or in association with viruses were identified in 45/57 (79%) cases including 34/40 (85%) SIDS. The most frequent bacterial isolate was Escherichia coli (27), and the virus identified most frequently was enterovirus (8). C-reactive protein was increased in 10 out of the 42 cases tested including 8/32 (25%) SIDS. Significant gastric content aspiration was found in 17/57 (29.8%) including 13/40 (32.5%) SIDS. Histological signs of shock were present in 33/55 (60%) cases including 22/39 SIDS (56.4%). The microbiological findings were positive for 27/33 (81.8%). We conclude that post-mortem microbiology is essential in sudden death investigation. The conclusion that a death is unexplained if no microbiology was done is not valid, even if in some cases it may be difficult to know precisely in what way the pathogen contributed to the death.