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.

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.

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.

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.

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.

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.

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.

[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.

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.

Seroepidemiological survey of toxoplasmosis in La Guadeloupe, French West Indies.

A random survey of 1% of the total population of La Guadeloupe, French West Indies, for toxoplasmosis was carried out. Blood specimens from 3,238 individuals were collected on filter paper strips and tested for Toxoplasma antibodies by the fluorescent antibody technique. Sixty percent were positive. A gradual increase in positivity with increasing age, and high prevalence in children (50% in the 6- to 10-year age group) were observed. No differences were found among the different ethnic groups, nor among persons in different occupations. Economic status showed a highly negative correlation with antibody prevalence rates. The infection rate was not higher in individuals who consumed raw or undercooked meat, and no differences in antibody prevalence related to the sources of meat were observed. The seropositivity rates varied over a wide range (40-76%) according to the locality; higher Toxoplasma antibody rates were found in areas with higher rainfall, and toxoplasmosis prevalence rates correlated positively with mean annual rainfall but not with altitude or with rural or urban residence. A significant difference was observed between seropositivity rates when people living in houses with or without cats were compared. A higher prevalence of hookworm and Strongyloides infections--considered as evidence of closer contact with moist soil--were found in children with Toxoplasma antibodies than in age-adjusted control children without such antibodies. These results provide evidence that meat plays a negligible role in the transmission of Toxoplasma to man, and support the hypothesis that oocysts shed in cat feces are the almost exclusive mode of human infection in Guadeloupe.

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.

Visceral leishmaniasis in southern Sudan.

Reports made by Médecins Sans Frontières in Khartoum on an outbreak of visceral leishmaniasis among displaced people from the western Upper Nile prompted an investigation at Ler Hospital, the second largest in the region. In a 10 d period during April 1989, 100 persons with visceral leishmaniasis were identified. Of these, 82% were men; 67% were aged 20 to 39 years. Except for the absence of ulcerated skin lesions, the clinical features corresponded to those traditionally described in the Sudan. A cross-sectional serological survey was conducted in Kuernyang (400 inhabitants), 40 km north of Ler. The anti-Leishmania antibody prevalence was 18.2%, being higher among those older than 15 years, and higher among adult women (28%) than among men (18%). The overall prevalence of splenomegaly was 16.4%. 33% of seropositive cases presented with splenomegaly, compared with 11.6% of those who were seronegative. Three serological surveys conducted on the eastern side of the Nile showed no seropositive cases. However, 2 autochthonous cases were clinically diagnosed and confirmed by serological assays. The war conflicts and population movements appear to be the main cause of this large outbreak that may have killed thousands of tribespeople in southern Sudan. There is a risk of the disease spreading into other areas with devastating consequences for the population, should energetic measures not be immediately taken.

Experimental trichinellosis in horses: biological and parasitological evaluation.

Three groups of three horses each were, respectively, infected with 5000, 20,000 and 50,000 larvae of Trichinella spiralis. The strain used was isolated from a human biopsy during horsemeat-related outbreaks of trichinellosis in France. Transient muscular disorders were only observed in two of the horses infected with 50,000 larvae but none of the horses had fever. A significant increase in blood eosinophils was noticed in 5 horses. Serum LDH, aldolase and CPK peaked at the fifth week post-infection. Specific IgG assayed by indirect immunofluorescence and ELISA, appeared 2-5 weeks post-infection and disappeared between 16 and 40 weeks. The distribution of T. spiralis larvae was maximal in the tongue, masseters and diaphragm, but a large decrease in the number of larvae recovered from the muscles was noticed among the horses slaughtered at the beginning and end of the experiment. In muscular histological sections, larvae were observed in an intramyofibrillar position and were surrounded by a mild to severe inflammatory reaction.

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

[Evaluation of the medical and social cost of 2 trichinosis outbreaks in France in 1985]. / Evaluation du coût médical et social de deux épidémies de trichinose survenues en France en 1985.

Two outbreaks of trichinosis occurred in France in 1985 and 1,073 cases were identified. These outbreaks were related to the consumption of parasitized horse meat. The medical and social cost of these outbreaks have been estimated with a sample of 92 patients. The average medical cost was 1,200 FF by outpatient and 17,300 FF by hospitalized patient. For the 1,073 cases, the total cost of these 2 outbreaks is estimated to have been between 7 and 11 millions FF. Of this sum, the hospitalization fees exceeded 51% and the social cost was 36% (loss of productivity). Since October 1985, a decree of the French Ministry of Agriculture has imposed a parasitologic test on all horse carcasses slaughtered in France or imported. This examination costs under 5 FF (under $1) per carcass. This study shows that the systematic examination for trichinosis of horse meat carcasses should be continued and that more sensitive screening methods should be developed.

Recent news on trichinellosis: another outbreak due to horsemeat consumption in France in 1993.

A new outbreak of trichinellosis occurred in France in December 1993 and involved around 550 patients. The authors report here how recent knowledge on Trichinella have been helpful to investigate this outbreak.

[2 epidemics of arsenical encephalopathy in the treatment of trypanosomiasis, Uganda, 1992-1993]. / Etude de deux épidémies d'encéphalopathies arsenicales dans le traitement de la trypanosomiase, Ouganda, 1992-1993.

Since 1988, the french non-governmental organisation Médecins Sans Frontières is running a control program of human african trypanosomiasis in the district of Moyo, North-Uganda. Between 1988 and 1993, more than 7,000 patients were diagnosed and treated. Since 1988, it has been noted that incidence of melarsoprol reaction had increased systematically between June and October of each year, indicating strong seasonal variation. In 1992 and 1993, two outbreaks of arsenical reactive encephalopathy (ARE) occurred in the sleeping sickness center of Adjumani. The incidence of ARE suddenly exceeded 10% of the patients treated by melarsoprol during August 1992 and September 1993. The onset of 80% of those "epidemic" cases, occurred between the 5th and the 11th day of treatment. Two retrospective studies were conducted in 1992 (75 cases) and in 1993 (51 cases). Among the risk factors studied, two appear to increase the risk of ARE: the prescription of thiabendazole to treat strongyloidiasis during the melarsoprol cure and the bad general clinical conditions of patients. These observations suggest that exogenous co-factors could be involved in the occurrence of ARE. Recommendations were to avoid administration of diffusible anti-helminthic treatment during the cure, and to improve the general conditions of patients before the cure of melarsoprol.

[Current aspects of medical mycology]. / Les aspects actuels de la mycologie médicale.

The authors tried in this article to review the principal aspects of deep mycoses. A special attention was paid to the clinical aspects of candidiasis, aspergillosis and cryptococcosis. The incidence of these diseases is now growing owing to the increasing use of immunosuppressives drugs and to AIDS. The difficulty of the biological diagnosis of these mycoses is discussed. Drugs currently used are amphotericin B, 5-fluoro-cytosine, ketoconazole and fluconazole. Prophylactic measures and the indications of the main drugs are considered.

Comparative value of polymerase chain reaction and conventional biological tests for the prenatal diagnosis of congenital toxoplasmosis.

When toxoplasmosis is acquired during pregnancy, there is a risk of severe congenital defect in the foetus. Maternal treatment with spiramycin limits the transplacental passage of the parasite to the foetus but does not prevent infection in all cases. Prenatal diagnosis should be based on specific and fast methods to prescribe the more potent combination of sulfadiazine and pyrimethamine. This study evaluates PCR in the prenatal diagnosis of toxoplasmosis; PCR was based on the detection of the gene coding for the P30 surface protein. Amniotic fluid from 44 women with suspected foetal infection was tested by PCR and results were compared to those of conventional diagnostic tests on foetal blood and amniotic fluid. PCR was positive in 7 out of 10 samples from proven congenital toxoplasmosis cases. Sensitivity of PCR was similar to cell culture and mouse inoculation of amniotic fluid but was superior to tests carried out on foetal blood (specific IgM, eosinophil and platelet counts, gamma glutamyl transferase, mouse inoculation). In two cases, PCR was positive with no detected infection of the foetus. In this study, the combination of fast detection methods, ie cell culture and PCR of amniotic fluid, eosinophil and platelet counts, GGT activity and specific IgM, enabled us to confirm 10/10 cases of congenital toxoplasmosis in less than a week. PCR therefore appears to be an additional test which improves early prenatal diagnosis of toxoplasmosis.