Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii.

Quantitative PCR (qPCR) is now a key diagnostic tool for Pneumocystis pneumonia. However, cutoffs to distinguish between infected and colonized patients according to their HIV status have not yet been determined. According to clinical, radiological, and biological data, we retrospectively classified bronchoalveolar lavage (BAL) samples subjected to qPCR over a 3-year period into four categories, i.e., definite PCP, probable PCP, Pneumocystis colonization, and no infection. Fungal burden was then analyzed according to the HIV status of the patients. Among 1,212 episodes of pneumonia screened in immunocompromised patients, 52 and 27 HIV-positive patients were diagnosed with a definite and probable PCP, whereas 4 and 22 HIV-negative patients had definite and probable PCP, respectively. Among patients with definite or a probable PCP, HIV-negative patients had a significantly lower burden than HIV-positive patients (P < 10(-4)). In both groups, the median fungal burden was significantly higher in patients with definite PCP than in colonized patients. A single cutoff at 1.5 × 10(4) copies/ml allowed to differentiate colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cutoff values of 2.87 × 10(4) and 3.39 × 10(3) copies/ml resulted in 100% specificity and sensitivity, respectively. Using cutoffs determined for the whole population would have led us to set aside the diagnosis of PCP in 9 HIV-negative patients with definite or probable PCP. qPCR appeared to be the most sensitive test to detect Pneumocystis in BAL samples. However, because of lower inocula in HIV-negative patients, different cutoffs must be used according to the HIV status to differentiate between colonized and infected patients.

Congenital toxoplasmosis in France in 2007: first results from a national surveillance system.

When immunocompetent people become infected with the parasite Toxoplasma gondii, the disease is generally asymptomatic. However, transplacental transmission of T. gondii may lead to severe congenital infection including in utero abortion, foetal death, or neurological or ocular damage of the foetus. France has had a national programme to prevent congenital toxoplasmosis since 1978. However, although estimated seroprevalence in pregnant women has fallen from 84% in the 1960s to 44% in 2003, no reliable data have been available on the annual number of cases of congenital toxoplasmosis or the severity of infection. In 2006, the French National Institute for Public Health Surveillance (Institut de Veille Sanitaire) and the National Reference Centre for Toxoplasmosis recommended that a national laboratory-based surveillance system be used for the surveillance of the disease. In 2007, 31 laboratories reported at least one congenital case through the surveillance system, giving a total of 272 cases. A total of 11 terminations of pregnancy were reported (six abortions and five foetal deaths). Of the live-born cases, 206 were asymptomatic, 28 were symptomatic and seven had a severe form of the disease. As there were 818,700 births in France and French overseas departments in 2007, the overall prevalence of congenital toxoplasmosis observed that year was 3.3 (95% confidence interval (CI): 2.9 to 3.7) per 10,000 live births and the incidence rate of the disease at birth was 2.9 (95% CI: 2.5 to 3.2) per 10,000 live births; the estimated incidence rate of symptomatic congenital toxoplasmosis was 0.34 (95% CI: 0.2 to 0.5) cases per 10,000 live births.

Evaluation of a rapid enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis.

Diagnosis of strongyloidiasis using stool examination remains unsatisfactory due to the lack of sensitivity and fastidious techniques. In this work, we investigated the value of an anti-Strongyloides IgG enzyme immunoassay (EIA), using a panel of 207 sera retrospectively collected from patients with definitive diagnoses of strongyloidiasis (n=57), other helminthic infections (n=46), eosinophilia without parasitic infection diagnosis (n=54), and digestive disturbances following a tropical journey (n=30) and from 20 negative controls. By following a receiver operating characteristic (ROC) curve analysis, it was possible to optimize the test to reach a sensitivity of 91.2% and a specificity of 93.3%, with 92.8% of patients correctly classified. Considering the incidence of strongyloidiasis diagnosed in our own laboratory, the negative predictive value was calculated at 99.9%. In conclusion, this test is very rapid and easy to perform and may be valuable for diagnosis of strongyloidiasis both in cases where the infection is unrevealed by a parasitological stool examination and in patients at risk for severe clinical forms, such as patients receiving immunosuppressive therapy.

Trichinellosis acquired in Nunavut, Canada in September 2009: meat from grizzly bear suspected.

Five cases of trichinellosis with onset of symptoms in September 2009, were reported in France, and were probably linked to the consumption of meat from a grizzly bear in Cambridge Bay in Nunavut, Canada. Travellers should be aware of the risks of eating raw or rare meat products in arctic regions, particularly game meat such as bear or walrus meat.

[How can the cost of screening for toxoplasmosis during pregnancy be reduced?]. / Comment réduire le coût du dépistage de la toxoplasmose chez la femme enceinte?

BACKGROUND: A program of systematic serology screening for toxoplasmosis during pregnancy has been running in France since 1978. The program involves monthly follow-ups for all non-immune pregnant women. Due to the steady decline in the seroprevalence of toxoplasmosis, the cost of the program is steadily increasing. Current screening is based on the detection of IgG and IgM isotypes. The aim of this work was to estimate the benefit of replacing combined dosage of two isotypes, by an alternative strategy that detects total anti-Toxoplasma immunoglobulins. METHODS: The rate of decreasing seroprevalence and the increasing burden on serological examinations was measured in a study population of pregnant women who were checked for toxoplasmosis by the parasitology laboratory of the Cochin Hospital, Paris. The increase in screening costs was estimated for the all-pregnant women and the expected benefits stemming from simply measuring total anti-Toxoplasma immunoglobulins compared to the double IgG-IgM assay were estimated. RESULTS: Between 1987 and 2008, the seroprevalence of toxoplasmosis measured at the Cochin hospital dropped from 70.8% to 48.6% with a 1.77% annual rate of decline. This downward trend is similar to that observed by the national perinatal surveys performed in 1995 and in 2003. As the number of non-immune women to follow-up each month is constantly increasing, the proportion of negative tests issued reached 87.6% in 2008. Extrapolating these results to the whole of France, we estimated that the number of required screening tests perform was increasing by 93,000 units per year with an additional associated cost of one million euros. Various alternative scenarios of antibody detection are proposed that could save between 40.2% and 48.4% of current screening costs. CONCLUSION: Replacement of combined dosage of IgG and IgM isotypes by determination of just total Ig would significantly reduce costs of toxoplasmosis screening for pregnant women, without effecting either the general strategy, or proven efficiency of the national program.

Multicenter comparative evaluation of five commercial methods for toxoplasma DNA extraction from amniotic fluid.

Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.

Trichinellosis acquired in Senegal from warthog ham, March 2009.

Three confirmed and three suspected cases of trichinellosis have been reported in France with onset of symptoms in March 2009, linked to consumption of smoked warthog ham in Senegal.

Contributions of immunoblotting, real-time PCR, and the Goldmann-Witmer coefficient to diagnosis of atypical toxoplasmic retinochoroiditis.

Ocular toxoplasmosis is a major cause of posterior uveitis worldwide. The diagnosis is based mainly on ophthalmological examination. Biological diagnosis is necessary in atypical cases, and this requires aqueous humor sampling by anterior chamber paracentesis. We evaluated real-time PCR targeting the Toxoplasma gondii 529-bp repeat element, the Goldmann-Witmer coefficient (GWC), and immunoblotting for the diagnosis of toxoplasmic retinochoroiditis in 54 patients with atypical uveitis. The results of these biological tests, applied to paired aqueous humor-serum samples, were compared to the clinical findings. Combining either PCR or the GWC with immunoblotting increased the sensitivity to 73% or 70%, respectively. Together, PCR and the GWC had 80% sensitivity. If feasible, sensitivity can be increased by combining the three methods (85% sensitivity). The interval between symptom onset and anterior chamber paracentesis strongly influenced the detection of specific intraocular antibody synthesis. The sensitivity of the GWC increased from 45% to 56% when sampling was performed 10 days after symptom onset, and that of immunoblotting increased from 53% to 72% when puncture was performed 30 days after symptom onset. PCR analysis of aqueous humor samples detected toxoplasmic DNA in 55% of patients. In contrast to the results of immunoblotting and the GWC, the results of PCR were not influenced by the interval between symptom onset and paracentesis. PCR was more informative than the GWC and immunoblotting for immunocompromised patients. Acute necrotizing retinal lesions were significantly larger in PCR-positive patients, with a mean of 3.5 optic disc diameters, than in PCR-negative patients, with a mean of 1.5 optic disc diameters.

[Antiviral immunization of immunocompromised adults, literature review]. / Vaccination antivirale des adultes immunodéprimés, revue de la littérature.

PURPOSE: Immunization, by preventing infections, has a major interest for the immunocompromised subjects. The aim of this article is to make a point on data concerning efficacy (in terms of immunogenicity) and safety of viral vaccines available in France and to synthesize existing guidelines for four groups of patients: solid organ and hematopoietic stem cell transplant recipients, HIV infected persons and patients treated by immunosuppressive drugs for a systemic disease. CURRENT KNOWLEDGE AND KEY POINTS: Available data about vaccines immunogenicity and safety for immunocompromised adults are rare. However, those data indicate that, when immunization contraindications and recommendations are applied, vaccines remain well tolerated and most of the time immunogenic, even if the percentage of responders is lower compared to non immunocompromised persons. Still, the specific guidelines that have been elaborated for immunization of immunocompromised adults are imprecise and incomplete, emphasizing a lack of data about this topic. FUTURE PROSPECTS: Clinical studies remain necessary to precise vaccines immunogenicity and safety for immunocompromised adults. In the meantime, a harmonization of immunization practices for immunocompromised adults should be proposed, so as to help practitioners to succeed a better immunization coverage for these patients.

Comparison of PCR, microscopic examination and culture for the early diagnosis and characterization of Acanthamoeba isolates from ocular infections.

In the study presented here, PCR, microscopic examination and culture of corneal samples were compared as methods of confirming the clinical diagnosis of Acanthamoeba keratitis, a serious ocular infection that is difficult to diagnose and threatens eyesight. The three methods were applied to isolates obtained from 513 patients with clinical signs or risk factors suggesting Acanthamoeba infection. Acanthamoeba keratitis was diagnosed in 12 of these patients. Combined PCR assays were more sensitive (94%) than either microscopic examination (33%) or culture (7%). The Acanthamoeba isolates were characterized using DNA sequence analysis of the nuclear small-subunit rRNA gene, and T4 was the predominant genotype found.

Application of microsatellite typing for the investigation of a cluster of cases of Candida albicans candidaemia.

A cluster of cases of Candida albicans candidaemia in a surgical intensive care unit was investigated. The probability of such a cluster during a single month was highly significant compared with the frequency of candidaemia in the previous year. A molecular typing method, based on length analysis of three (EF3, CDC3, HIS3) microsatellite-containing regions, was used to investigate isolates from patients in and outside the ward. This demonstrated the involvement of different strains, indicating the absence of cross-transmission among patients. Results of microsatellite typing can be obtained almost in real-time, which is particularly useful in an outbreak context.

Foodborne infections in France.

To quantify the impact of foodborne diseases on health, and set priorities for data collection, prevention and control of these diseases, we compiled and analyzed information from surveillance systems and other sources on the morbidity and mortality due to foodborne infectious diseases in mainland France in the last decade of the 20th century. Illness due to 13 bacteria, two viruses, and eight parasites were studied. The number of foodborne infections, hospitalizations, and deaths were estimated from multiple data sources. For each agent, several estimates were derived from the different sources. Estimates were ranked according to their plausibility, based on an assessment of the validity of the data source, and are presented as a "plausible interval" consisting of a low and high estimate. We estimate that these pathogens caused 10,200-17,800 hospitalizations per year. Salmonella is the most frequent cause (5,700-10,200 cases), followed by Campylobacter (2,600-3,500 cases) and Listeria (304 cases). Toxoplasmosis accounts for the majority of hospitalizations (426 cases) attributable to the studied parasitic infections. The number of deaths related to foodborne infection was estimated between 228 and 691. Bacterial pathogens account for the majority (191 to 652) of deaths of which 92 to 535 are attributable to salmonellosis, ranking as the first cause of death, and 78 to listeriosis, the second cause. Salmonella, Campylobacter, and Listeria are the main causes of severe foodborne illness in France. For several pathogens, data are insufficient to derive exact estimates of the disease burden. Nevertheless, it has been possible to derive plausible estimates for the majority, and to rank them according to their impact on public health.

Comparison of aerobic standard medium with specific fungal medium for detecting fusarium spp in blood cultures.

In order to compare the performances of a specific fungal medium and a standard aerobic medium for detecting growth of Fusarium spp. in blood, simulated blood cultures were performed. For lower inocula (10(2) and 10(3) cfu/ml taken together), fungal growth was detected significantly earlier using the fungal medium. The mean difference in the time to detection between the two media was 22.33 h at 10(2) cfu/ml, with the maximum difference being achieved for Fusarium verticilloides at 37.05 h. These in vitro test results suggest fungal medium could be useful for obtaining more rapid blood culture results when evaluating patients at risk for invasive infection with Fusarium spp.

Risk factors for Toxoplasma infection in pregnancy: a case-control study in France.

Each year an estimated 4900 cases of primary Toxoplasma infection occur in pregnant women in France, a country with a high prevalence. Since 1992 all pregnant women at risk of Toxoplasma infection have been required to undergo monthly serological testing. This case-control study, the first of its kind in France, was undertaken to identify risk factors for Toxoplasma infection during pregnancy, with a view to improving primary prevention among non-immune pregnant women. A total of 80 pregnant women who seroconverted to Toxoplasma were matched with 80 pregnant women who had repeatedly negative tests. The women were interviewed by telephone, using a standardized questionnaire, to determine socio-demographic characteristics, exposure to possible risk factors and the type of information on prevention received during pregnancy. The risk factors for Toxoplasma infection included in a multivariate analysis were poor hand hygiene (OR = 9.9; 95%CI: 0.8-125), consumption of undercooked beef (OR = 5.5; 95%CI: 1.1-27), having a pet cat (OR =4.5; 95%CI: 1.0-19.9), frequent consumption of raw vegetables outside the home (OR = 3.1; 95%,CI: 1.2-7.7) and consumption of undercooked lamb (OR = 3.1; 95%CI: 0.85-14). Receipt of documentary advice on prevention was associated with a lower risk of infection. Prevention campaigns among pregnant women in France could be improved and should focus on eating habits, hand hygiene and cats.

Value of prenatal diagnosis and early postnatal diagnosis of congenital toxoplasmosis: retrospective study of 110 cases.

We reviewed the files of 110 women with Toxoplasma seroconversion during pregnancy. Prenatal diagnosis was attempted for 94 women by amniotic fluid sampling. Toxoplasma gondii was detected by PCR, with or without tissue culture and mouse inoculation. The early neonatal diagnostic procedure included placental testing by PCR and/or mouse inoculation, cord blood serological testing, and comparison of maternal and newborn antibodies by Western blotting (WB). Serological follow-up of the infants was conducted during the first year of life or until the diagnosis of congenital toxoplasmosis (CT) could be ruled out. Congenital infection was diagnosed in 27 individuals (20 live births) in the prenatal and/or neonatal period. The sensitivity and specificity of prenatal diagnosis were 81 and 100%, respectively. Placental examination was positive for 66.7% of individuals with CT and was always negative for neonates without CT. Cord blood serology detected immunoglobulin M (IgM) and/or IgA in 80% of infected newborns, with respective specificities of 91.2 and 87.7%. By WB we detected bands on IgG and IgM blots recognized by the newborn serum but not by the maternal serum (neosynthesized IgG and/or IgM) for 88.2% of infected infants within the first 2 months of life with a specificity of 100%. Early postnatal diagnosis was negative for 2 of the 20 neonates with CT. Both of these newborns had a negative prenatal diagnosis and were asymptomatic, suggesting a very low parasite load. In conclusion, despite the use of advanced methods, some cases of congenital toxoplasmosis cannot be detected early, which underlines the importance of careful follow-up of newborns who are at risk.

A multifocal outbreak of trichinellosis linked to horse meat imported from North America to France in 1993.

An outbreak of 538 cases of trichinellosis occurred in France in December 1993. Seven cases developed neurotrichinosis and 23 had cardiologic complications. No deaths were recorded. Two patients had a positive muscle biopsy showing living Trichinella larvae. One of them was typed as Trichinella spiralis. A case-control study showed that horse meat was the only meat associated with illness (odds ratio = 80.7). The risk of illness increased with the amount of horse meat eaten and when it was consumed raw. The cases, which were spread out in five foci, bought horse meat from five butchers who had received parts of a single horse carcass imported in November 1993 from Canada. The Trichinella International Screening Program, implemented since 1985 after two similar episodes involving a thousand cases, failed to detect the incriminated horse carcass. This new horse meat-related outbreak led to modifications of the internationally recommended screening methods whereby the weight of meat samples tested was increased.

History of trichinellosis outbreaks linked to horse meat consumption 1975-1998.

Trichinellosis is a parasitic disease transmitted to humans by consumption of raw or undercooked meat from animals contaminated by worms of the Trichinella genus. In nature, the life cycle involves most carnivores, omnivores, and numerous rodents. Domesti