Maternal administration of valaciclovir in symptomatic intrauterine cytomegalovirus infection.

OBJECTIVES: To report early experience with treatment of intrauterine cytomegalovirus (CMV) infection using maternal oral administration of valaciclovir (VACV). DESIGN: Observational study of fetuses infected with CMV with or without treatment with valaciclovir. POPULATION: Pregnancies with confirmed fetal CMV infection were treated with oral VACV (8 g/day). MAIN OUTCOME MEASURES: Fetal viral load and drug concentration were monitored in amniotic fluid and in fetal blood. Data on the course and outcome of a group of untreated symptomatic fetuses infected with CMV are also reported. RESULTS: Therapeutic concentrations were achieved in maternal and fetal bloods. The viral load in the fetal blood (VLFB) decreased significantly after 1-12 weeks of treatment (Wilcoxon paired test P = 0.02). Twenty pregnancies including 21 fetuses were treated at 28 weeks (median, range: 22-34) for 7 weeks (median, range: 1-12). Ten infants were developing normally at between 1 and 5 years of age. Two infants (both aged 2 years) had severe isolated unilateral deafness. One neonate presented with microcephaly and severe deafness but was also diagnosed with incontinentia pigmenti. Six out of seven cases that eventually required termination of pregnancy (TOP) had evidence of in utero progression of the disease with worsening cerebral lesions. One fetus died in utero. The outcome of 14/24 (58.3%) untreated symptomatic infected fetuses was poor with either TOP, intrauterine fetal demise or severe congenital infection disease of the neonate; the remaining ten infants were healthy at follow up. CONCLUSION: Maternal oral administration of VACV leads to therapeutic concentrations in the maternal and fetal compartments, with a decrease in VLFB. Our results suggest that in cases where TOP is declined, a randomised controlled trial to study this treatment option further is indicated.

Necrotising retinopathies simulating acute retinal necrosis syndrome.

AIM: To determine an aetiological diagnosis in patients presenting with necrotising retinopathies that simulate acute retinal necrosis (ARN). METHODS: Retrospective non-comparative case series. The charts of 16 patients presenting with a clinical impression of ARN at Pitie-Salpetriere Hospital, Paris, France, between 1994 and 1999, who required initial antiviral therapy were reviewed. All of the patients had extensive laboratory tests. Anterior chamber paracentesis was performed on 14 patients and evaluated by polymerase chain reaction (PCR) and/or the Witmer-Goldmann coefficient to determine the cause of retinitis. Three of the 14 cases also had diagnostic vitrectomy. Responses to specific treatment, initiated based on laboratory results, and the final outcome were evaluated. RESULTS: Seven of the 16 patients were female and nine were male. The retinitis was bilateral in five patients and unilateral in 11 patients. The average age of the patients at presentation was 53.6 years. 13 patients were immune deficient for various reasons. Upon initial presentation, the patients' visual acuities were less than 20/200 in 68% of the affected eyes. The final diagnoses, based on laboratory data and therapeutic response were toxoplasmic retinochoroiditis (62.5%), syphilitic retinitis (12.5%), aspergillus endophthalmitis (12.5%), Behcet's disease (6.2%), and intraocular lymphoma (6.2%). Visual acuity was stabilised or improved in 12 patients (75%). Two patients with aspergillosis died despite antifungal therapy. CONCLUSIONS: Toxoplasmic retinochoroiditis is the major cause of retinal necrosis that simulates ARN, and PCR analysis of the aqueous humour is helpful in establishing the diagnosis. Such atypical toxoplasma retinochoroiditis may be associated with poor visual outcome.

Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt.

Sera from 75 water buffaloes from Egypt were examined using a direct agglutination test incorporating mercaptoethanol for antibodies to Neospora caninum and Toxoplasma gondii. Antibodies to N. caninum were found in 51 (68%) of 75 buffaloes in titres of 1:20 (six buffaloes), 1:40 (15 buffaloes), 1:160 (one buffalo), 1:320 (one buffalo) and > or = 1:640 (28 buffaloes), using N. caninum formalin-preserved whole tachyzoites as antigen. Antibodies to T. gondii were not found in a 1:100 dilution of serum of any of the 75 buffaloes, using T. gondii as antigen, indicating specificity in the detection of antibodies to N. caninum. This is the first report of N. caninum prevalence in water buffaloes, which are economically very important domestic animals in developing countries.

High prevalence of antibodies to Neospora caninum in white-tailed deer (Odocoileus virginianus).

Serum samples of 400 white-tailed deer (Odocoileus virginianus) from 16 preserves in northeastern Illinois were tested for Neospora caninum antibodies in the N. caninum agglutination test using mouse-derived N. caninum tachyzoites and mercaptoethanol. Antibodies were found in 162 deer with titres of 1:40 (47 deer), 1:80 (32 deer), 1:160 (17 deer), 1:200 (eight deer), 1:400 (19 deer), 1:800 (17 deer) and > or = 1:1600 (22 deer). There were no significant differences in prevalence between age or sex of the deer. The high prevalence of N. caninum infection in deer is consistent with a sylvatic cycle of N. caninum.

Prenatal diagnosis using polymerase chain reaction on amniotic fluid for congenital toxoplasmosis.

OBJECTIVE: To evaluate sensitivity, specificity, and predictive values of a prenatal amniotic fluid (AF) polymerase chain reaction (PCR) test for diagnosis of congenital toxoplasmosis. METHODS: A multicenter prospective study was done on 271 women with proved primary Toxoplasma infection during pregnancy and who had amniocentesis for prenatal diagnosis by PCR. Live-born infants were eligible for analysis only if a serologic follow-up could assess a definitive infection status. RESULTS: Of the 270 evaluable cases, 75 were congenitally infected, 48 of whom had a positive PCR at prenatal diagnosis. Overall sensitivity of PCR on AF was estimated at 64% (95% confidence interval [CI] 53.1%, 74.9%), negative predictive value of 87.8% (95% CI 83.5%, 92.1%), whereas specificity and positive predictive value were 100% (95% CIs 98%, 100% and 92.3%, 100%, respectively). Among cases with congenital toxoplasmosis, there were no significant differences between those with positive or negative PCR with regard to median gestational age at maternal infection, interval between maternal infection and amniocentesis, or duration of treatment before amniocentesis. However, sensitivity of PCR was found to be significantly higher for maternal infections that occurred between 17 and 21 weeks' gestation (P <.02). CONCLUSION: A negative PCR of AF cannot rule out congenital infection. In this case, continuation of treatment with spiramycin combined with ultrasonographic follow-up and postnatal follow-up are warranted. Our results also suggest presumptive treatment combining pyrimethamine and sulfonamides in case of maternal infection occurring late in pregnancy.

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in sera from camels from Egypt.

Sera from camels from Egypt were examined by the direct agglutination tests incorporating mercaptoethanol for antibodies to Neospora caninum and Toxoplasma gondii. Antibodies to N. caninum were found in 6 of 161 camels in titers of 1:40 (2 camels) and 1:80, 1:160, 1:640, and 1:1280 in 1 camel each, using N. caninum formalin preserved whole tachyzoites as antigen. Antibodies to T. gondii were found in 17.4% of 166 camels in titers of 1:25 (3 camels), 1:50 (18 camels). and > 1:500 (8 camels) using T. gondii tachyzoites. All 6 camels with N. caninum antibodies had no T. gondii antibodies in 1:4 dilution of serum, indicating specificity of the reaction. This is the first report of N. caninum prevalence in Egypt.

Serologic prevalence of Toxoplasma gondii in horses slaughtered for food in North America.

Serum samples from 1788 horses slaughtered for food in North America were tested for antibodies to Toxoplasma gondii using the modified direct agglutination test (MAT). Antibodies to T. gondii were found by the MAT in 124 (6.9%) of 1788 sera; the titers were 1:20 (69 horses), 1:40 (37 horses), 1:80 (9 horses), and > or =1:160 (9 horses). A total of 339 selected horses were also tested by the Sabin-Feldman dye test (DT). Dye test antibodies were found in 54 horses with titers of 1:10 (29 horses) 1:20 (12 horses), 1:40 (4 horses) and 1:80 (9 horses). There was no correlation between the DT and the MAT.

Serological investigation of an outbreak of Neospora caninum-associated abortion in a dairy herd in southeastern United States.

An outbreak of Neospora caninum-associated abortion occurred in a South Carolina dairy wherein greater than 10% of the herd aborted over a 4-month period. Of the total number of cows at mid-late gestation, nearly 40% (28/71) aborted while the remaining 60% (43/71) gave birth to normal calves. Immunohistochemical examination of brain tissue from a subset of aborted fetuses confirmed N. caninum as the causative agent of abortion in these animals. A variety of serological assays, including indirect fluorescence antibody test (IFAT), recombinant enzyme-linked immunosorbent assay (rELISA), ISCOM-ELISA, avidity ELISA, and Neospora agglutination test (NAT), were used to evaluate sera collected during the outbreak from 240 cows for antibodies to N. caninum. IFAT and ISCOM-ELISA testing showed that nearly 80% of the dairy cows had antibodies to N. caninum. NAT and rELISA had similar levels of seropositivity relative to IFAT and ISCOM-ELISA, but the percentage of positive sera was dependent on the cut-off value chosen. As indicated by kappa coefficient statistical analysis, ISCOM-ELISA and IFAT exhibited the highest level of agreement in identifying N. caninum-positive and -negative cows. A decrease in the percentage of seropositive cows as determined by ISCOM-ELISA and IFAT with increasing cow age was noted. However, no significant difference was observed between cow age and abortion status. In addition to these tests, an avidity ELISA was performed on all sera with high (> or =0.4) ISCOM-ELISA readings. Avidity index (AI) increased with time post-abortion suggesting that most abortions were due to recent N. caninum infection. Of the cows at risk for abortion, the mean serological AI of aborting cows was significantly lower (P<0.05) than mean serological AI of non-aborting cows.

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis.

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and Sarcocystis canis-like infections in marine mammals.

Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, and S. canis are related protozoans that can cause mortality in many species of domestic and wild animals. Recently, T. gondii and S. neurona were recognized to cause encephalitis in marine mammals. As yet, there is no report of natural exposure of N. caninum in marine mammals. In the present study, antibodies to T. gondii and N. caninum were assayed in sera of several species of marine mammals. For T. gondii, sera were diluted 1:25, 1:50, and 1:500 and assayed in the T. gondii modified agglutination test (MAT). Antibodies (MAT > or =1:25) to T. gondii were found in 89 of 115 (77%) dead, and 18 of 30 (60%) apparently healthy sea otters (Enhydra lutris), 51 of 311 (16%) Pacific harbor seals (Phoca vitulina), 19 of 45 (42%) sea lions (Eumetopias jubatus) [corrected] 5 of 32 (16%) ringed seals (Phoca hispida), 4 of 8 (50%) bearded seals (Erignathus barbatus), 1 of 9 (11.1%) spotted seals (Phoca largha), 138 of 141 (98%) Atlantic bottlenose dolphins (Tursiops truncatus), and 3 of 53 (6%) walruses (Odobenus rosmarus). For N. caninum, sera were diluted 1:40, 1:80, 1:160, and 1:320 and examined with the Neospora agglutination test (NAT) using mouse-derived tachyzoites. NAT antibodies were found in 3 of 53 (6%) walruses, 28 of 145 (19%) sea otters, 11 of 311 (3.5%) harbor seals, 1 of 27 (3.7%) sea lions, 4 of 32 (12.5%) ringed seals, 1 of 8 (12.5%) bearded seals, and 43 of 47 (91%) bottlenose dolphins. To our knowledge, this is the first report of N. caninum antibodies in any marine mammal, and the first report of T. gondii antibodies in walruses and in ringed, bearded, spotted, and ribbon seals. Current information on T. gondii-like and Sarcocystis-like infections in marine mammals is reviewed. New cases of clinical S. canis and T. gondii infections are also reported in sea lions, and T. gondii infection in an Antillean manatee (Trichechus manatus manatus).

Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases.

Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or stayed in the US exhibited titers over 50, a higher figure than in the group which did not travel out of France or stayed in an other European country. No correlation was found between anti-S. neurona and anti-Neospora sp. antibody titers. Data prompt further study of significance of anti-S. neurona antibodies in clinically healthy or diseased European horses, and identification of putative immunizing parasite(s) and their host(s).

Prevalence of antibodies to Neospora caninum in horses in North America.

Serum samples from 296 horses slaughtered for food in the United States were tested for antibodies to Neospora caninum by the Neospora-agglutination test (NAT). Antibodies were found in 69 (23.3%) horses with titers of 1:40 (19 horses), 1:80 (19 horses), 1:100 (3 horses), 1:200 (7 horses), 1:400 (4 horses), and 1:800 (17 horses). This is the first serologic survey for N. caninum antibodies in horses.

High prevalence of viable Toxoplasma gondii infection in market weight pigs from a farm in Massachusetts.

The ingestion of uncooked infected meat is considered important in the epidemiology of Toxoplasma gondii infection in humans and little is known of the prevalence of viable T. gondii in meat used for human consumption in the United States. In the present study, viable T. gondii was isolated from 51 out of 55 pigs destined for human consumption. Hearts and tongues (500 g) from fifty-five 6-mo-old pigs from a farm in Massachusetts were bioassayed for T. gondii by feeding them to T. gondii-free cats. Feces of these cats were examined for shedding of T. gondii oocysts. Fifty-one of 55 cats fed pig tissues each shed 25-810 million T. gondii oocysts in their feces. Two of these cats consumed tissues of pigs that were shown to be seronegative with the Sabin-Feldman dye test, the modified agglutination test, and the Western blot. Results indicate that until examination of meat for T. gondii infection is implemented in slaughterhouses, all meat should be cooked according to industry guidelines before human consumption.

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming.

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.

Prevalence of antibodies to Neospora caninum and Sarcocystis neurona in sera of domestic cats from Brazil.

Parasite Biology, Epidemiology and Systematics Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 1001, Beltsville, Maryland 20705-2350 Antibodies to Neospora caninum and Sarcocystis neurona were determined in serum samples of 502 domestic cats from Brazil using direct agglutination tests with the respective antigens. Antibodies to S. neurona were not found in 1:50 dilution of any serum in the S. neurona agglutination test. suggesting that domestic cats from São Paulo city were not exposed to S. neurona sporocysts from opossums. Antibodies to N. caninum were found in 60 (11.9%) of 502 cats with titers of 1:40 in 36 cats, 1:80 in 18 cats, 1:160 in 5 cats, and 1:800 in 1 cat using the Neospora agglutination test (NAT). Antibodies to N. caninum were confirmed by Western blotting in the sera of 10 cats with NAT titers of 1:80 to 1:800; this finding suggests that at least 10 cats had N. caninum-specific antibodies confirmed by 2 tests. This is the first documentation of natural exposure of cats to N. caninum.

Factors associated with microsporidial and cryptosporidial diarrhea in HIV infected patients.

Cryptosporidium and microsporidia are increasingly recognized as important agents of chronic diarrhea in human immunodeficiency virus (HIV) infected patients. These protozoa present clinical and biological similarities but coinfection with these two parasites seems uncommon in a population of diarrheic HIV infected patients in the Paris area (France), a comparison study was performed in order to clarify epidemiological differences between these protozoa. From November 1993 to December 1994, 26 microsporidial infected patients were compared to 28 cryptosporidial patients for various factors. Results of a multivariate logistic regression analysis showed that trips to tropical countries remained strongly associated with microsporidic compared with Cryptosporidium adjusted odds ratio [OR] = 4.6, 95% confidence interval [CI] 1.1-19.5). Thus, as compared with cryptosporidiosis, specific epidemiological factors could be associated with microsporidial transmission in tropical countries.

[Experimental models of toxoplasmosis. Pharmacological applications]. / Modèles expérimentaux de toxoplasmose. Applications pharmacologiques.

Toxoplasma gondii is an ubiquitous protozoan parasite causing severe or life-threatening infections in immunocompromised patients and in congenitally infected infants. Animal models have been extensively used to describe the pathology of infection and to identify new effective drugs for the treatment of congenital infections, chrorioretinitis and toxoplasmic encephalitis. Although inherent differences between man and animal can reduce the relevance of data obtained experimentally, animal models have greatly improved our knowledge on the various aspects of toxoplasmosis. Toxoplasma infection can be easily obtained in most laboratory animals, with exception of rats which are partially resistant. According to the strain used, the resulting infection may be acute, subacute or chronic, and can be monitored either by the survival of animals, the histopathological examination of lesions or, preferably, by titration of parasites in infected tissues using subinoculation to mice or tissue culture. This latter method has proved particularly useful to describe the kinetics of infection in host tissues and to assess the efficacy of drugs, according to their pharmacokinetics and tissue distribution. The relevance of results obtained in animal models of congenital toxoplasmosis and of chrorioretinitis is more questionable, due to the marked differences between the mode of infection in humans and in animals. Experiments performed in primates provided valuable informations for the management of therapy of congenital toxoplasmosis but were of limited interest for ocular toxoplasmosis. The pathogeny of toxoplasmic encephalitis is still poorly understood, and no experimental model is fully satisfactory to produce focal encephalitic lesions as observed in immunocompromised humans. Acute infections with highly virulent strains induce disseminated infection with major pulmonary and brain involvement, and thus can be used to assess the efficacity of drugs in these tissues. Direct inoculation of tachyzoites into brain tissue can induce focal encephalitis but this model is of difficult use for large scale studies. Although cellular immunity is mainly responsible for the control of toxoplasmosis at the chronic stage, administration of immunosuppressive drugs does not usually result in focal brain reactivation; such reactivation can only be obtained using antibodies against CD8 and CD4 T lymphocytes or interferon gamma. Another experimental approach is the use of genetically immunodeficient animals: these models are of limited interest for pharmacological research since infection of nude or T depleted mice usually results in a dissemination of infection; however, using these models it could be clearly demonstrated that immunity plays a major adjunctive role in the control of acute infection. Concurrent infections between viruses and parasites is a common feature in immunocompromised patients and especially during AIDS.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of Toxoplasma gondii parasitemia by polymerase chain reaction in perorally infected mice.

Sequential blood samples collected from mice infected perorally with an avirulent strain of T. gondii were analysed for parasite DNA by a polymerase chain reaction method (PCR). Two pairs of primers specific for gene B1 and the repetitive DNA sequence TGR1E were used for DNA amplification. Amplified products were detected by means of electrophoresis with ethidium bromide staining. Parasitemia was also determined by cell culture. Parasitemia was never detected by the tissue culture method, whereas parasite DNA was continuously detected with PCR from day 2 to day 21. These results confirm the high sensitivity of PCR for T. gondii DNA in blood, and show that circulating DNA is present for long periods in mice following primary infection.

[Intestinal bilharziasis with rectal bleeding: apropos of 3 cases]. / Bilharziose intestinale avec rectorragies: à propos de trois cas.

The authors report three cases of Schistosoma mansoni infection among European women coming back from Burkina-Faso. Six weeks after the infestation, these three patients showed, at first, a bloody diarrhoea, then large oedemas. The treatment, with praziquantel, was not tolerate very well and all the three patients had a high fever after. This case report requests a clarification about the frequency of such symptoms. The authors focus, through this observation, on the necessity of increasing the number of stools parasitologic analysis in order to diagnose. A sole negative analysis cannot eliminate it. Sometimes, only the rectal biopsy can find the eggs of the parasite.

[Extensive toxoplasmic retinochoroiditis. Diagnostic and therapeutic management]. / Toxoplasmose oculaire extensive. Conduite diagnostique et thérapeutique.

INTRODUCTION: To assess the diagnostic and therapeutic management of extensive toxoplasmic retinochoroiditis. PATIENTS AND METHODS: The files of all patients referred between December 1999 and December 2001 for the management of a severe, potentially sight-threatening toxoplasmic retinochoroiditis were retrospectively analyzed. The therapeutic strategy and the progression of intraocular inflammation are reported. RESULTS: Thirteen eyes of seven patients were finally included in the study. The sex ratio (F/M) and the mean age were respectively 4/3 and 44.5 years. Most of the patients were immunocompromised. Both eyes were initially affected in five cases. The diagnosis was confirmed by polymerase chain reaction (PCR) after anterior chamber paracentesis in six cases. Retinal detachment was observed in three cases, initially or during follow-up. All patients were treated with a combination of sulfadiazine and pyrimethamine, but azithromycin was necessary in two cases. Clindamycin was used in two cases of allergy to sulfadiazine. Corticosteroids were associated in five cases. For all patients, infection and inflammation were finally controlled. The visual acuity improved more than two lines in four eyes and remained stable in seven other eyes. DISCUSSION: Clinical diagnosis is still a challenge in severe cases of extensive toxoplasmic retinochoroiditis. PCR is helpful in identifying Toxoplasma gondii DNA. A systemic immunosuppression is frequently associated with a positive PCR. Treatment is based on a standard antiparasitic association and steroids must be discussed for each case according to the intensity of inflammation and the degree of immunosuppression. CONCLUSION: Extensive ocular toxoplasmosis is a serious condition. The final prognosis depends on the location of the necrotic lesions, rapid diagnosis, and efficient treatment.