Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP analysis

Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR-B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.     

Performance of Polymerase Chain Reaction Analysis of the Amniotic Fluid of Pregnant Women for Diagnosis of Congenital Toxoplasmosis: A Systematic Review and Meta-Analysis

IntroductionCongenital infection caused by Toxoplasma gondii can cause serious damage that can be diagnosed in utero or at birth, although most infants are asymptomatic at birth. Prenatal diagnosis of congenital toxoplasmosis considerably improves the prognosis and outcome for infected infants. For this reason, an assay for the quick, sensitive, and safe diagnosis of fetal toxoplasmosis is desirable.GoalTo systematically review the performance of polymerase chain reaction (PCR) analysis of the amniotic fluid of pregnant women with recent serological toxoplasmosis diagnoses for the diagnosis of fetal toxoplasmosis.MethodA systematic literature review was conducted via a search of electronic databases; the literature included primary studies of the diagnostic accuracy of PCR analysis of amniotic fluid from pregnant women who seroconverted during pregnancy. The PCR test was compared to a gold standard for diagnosis.ResultsA total of 1.269 summaries were obtained from the electronic database and reviewed, and 20 studies, comprising 4.171 samples, met the established inclusion criteria and were included in the review. The following results were obtained: studies about PCR assays for fetal toxoplasmosis are generally susceptible to bias; reports of the tests’ use lack critical information; the protocols varied among studies; the heterogeneity among studies was concentrated in the tests’ sensitivity; there was evidence that the sensitivity of the tests increases with time, as represented by the trimester; and there was more heterogeneity among studies in which there was more time between maternal diagnosis and fetal testing. The sensitivity of the method, if performed up to five weeks after maternal diagnosis, was 87% and specificity was 99%.ConclusionThe global sensitivity heterogeneity of the PCR test in this review was 66.5% (I²). The tests show low evidence of heterogeneity with a sensitivity of 87% and specificity of 99% when performed up to five weeks after maternal diagnosis. The test has a known performance and could be recommended for use up to five weeks after maternal diagnosis, when there is suspicion of fetal toxoplasmosis.     

Clinical presentation and diagnosis of toxoplasmic encephalitis in Japan

Distinguishing life-threatening toxoplasmic encephalitis (TE) from brain lymphoma in patients with acquired immunodeficiency syndrome (AIDS) may be difficult. Empiric anti-toxoplasmosis treatment is often initiated because of the reluctance in performing brain biopsies, which may delay the diagnosis and treatment of brain lymphoma in Japan. In this study, we retrospectively examined the clinical characteristics of 13 AIDS patients with TE in Japan, including magnetic resonance imaging and thallium 201 (201TI) single photon emission computed tomography (SPECT) findings, cerebral spinal fluid analysis, serology, and polymerase chain reaction (PCR) results. All patients improved on anti-toxoplasmosis treatment. Of the 11 patients who underwent serological testing, 6 (55%) had a positive serological result. Of the 7 patients who underwent PCR testing, 3 (42.9%) had a positive PCR result. Nine of 11 patients with TE (81.8%) had multiple lesions. Analysis of the sites of TE lesions did not reveal a difference in site predilection between TE and brain lymphoma. Uptake was negative in all 9 patients who underwent 201Tl SPECT. The study findings suggest that toxoplasma serostatus and PCR may be used to discriminate TE from brain lymphoma. No focal accumulation of 201TI is strongly suggestive of TE in patients with AIDS in Japan.     

Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals

Diagnosis of toxoplasmosis is useful for human and animal health. Several techniques are employed for the diagnosis in feline and canine population. Coprological tests for the detection of oocysts in cat faeces are of little significance owing to short patency (15 days). Histological examinations of biological samples show a lack of reliability when the animals are infected with few parasites; the mouse inoculation is the most reliable method even if the detection of cysts in mice brain require 40 days. However tachyzoites of virulent strains can be isolated from peritoneal exudate 3-4 days after inoculation. Samples inoculation in cell cultures (VERO, human fibroblasts) requires specialized laboratories and fails if non viable parasites are present due to tissutal autolysis. Serological tests are the most used diagnostic methods; Dye test and IFAT that require intact tachyzoites are more sensitive and specific compared to IHA, LA, ELISA because, during the infection, the first significant increase of IgM and IgG antibodies was observed against cuticolar antigens. A PCR to identify T. gondii DNA in canine and feline biological samples was developed. The B1 PCR performed on blood samples was less sensitive than when it was performed on other biological fluids requiring 100 tachyzoites, instead of 10. Aqueous humor PCR results could be negative if the infection is low grade or is restricted to the posterior segment or the animal was previously treated with anti-Toxoplasma drugs. SNC disease may be also difficult to diagnose because an high serum IgG titer may be associated with locally production or leakage from serum through a compromised blood-CSF barrier. AB1 PCR was successfully applied for the diagnosis of Toxoplasma abortion in ewes requiring only 10 parasites in placental cotyledon samples; the test compared with mouse inoculation showed similar sensitivity. Discrepancies may have been due to a low and focal distribution of parasites in the tissues or to the presence of non viable parasites if the tissues are autolysed. In regard to diagnostic methods adaptable to slaughter testing, several serological tests have been studied (IFAT, ELISA, IHA) for detection of IgG in sheep, pigs, cattle using also recombinant antigens (gene fragments H4 and H11) to lack the cross reactivity. The problem is the antibodies fall to near background levels as the infection became chronic (6-10 months p.-i.). A highly sensitive and specific method (Toxo Taq Man) has been developed to detect and quantitate T. gondii burden in animal tissue samples (0.1 pg of T. gondii genomic DNA, which is equivalent to 1 bradyzoite) using T. gondii ITS1-derived primers and a fluorogenic probe via Real-Time PCR. This assay is compatible with automation technology for potential slaughterhouse use. The diagnosis of acute infection in human pregnancy is difficult since IgM antibodies can be detected for a very long time after the acute phase; an IgA increase is of more diagnostic value because can be detected only for 6-7 months while the short kinetics of IgE can be useful only to date the infection precisely. In addition an IgG seroconversion is essential for the diagnosis. Among the most reliable tests, IgG avidity test is useful when a single serum sample, in the first months of gestation, is available, but low avidity results may persist for as long as 1 year. For this purpose a panel of serologic tests must be performed (ELISA, EIA, ISAGA, IgG avidity, IFAT, Dye test) for IgM, IgA, IgG and IgE. The serological diagnosis of prenatal infection is difficult since maternal IgG are passively transferred in utero to the foetus and caution must be exercised in interpretation of IgM or IgA results. A technique of Western blots of paired maternal and baby sera for evidencing different bands in the blots of two sera was developed for this purpose (specificity 97-100%, sensitivity 96-98%). The most reliable methods for prenatal diagnosis are PCR, mouse inoculation and cultural techniques performed on amniotic fluid, foetal blood and peripheral maternal blood in pregnants serologically positive. PCR (targets B1, SAG-1, rDNA) with amniotic fluid performed from 18 weeks of gestation is more sensitive and more rapid than conventional diagnostic procedures. PCR has been successfully used to diagnose Toxoplasma encephalitis in immunocompromised patients (cerebral biopsy is the only diagnostic method) and in ocular toxoplasmosis. In this evenience it is useful the study of IgG, IgM, IgA profile of paired serum and aqueous humor (Western blots).     

Delayed Cerebral Toxoplasmosis in a Kidney Transplant Patient: a Case Report

Cerebral toxoplasmosis is often life-threatening in an immunocompromised patient due to delayed diagnosis and treatment. Several differential diagnoses could be possible only with preoperative brain images of cerebral toxoplasmosis which show multiple rim-enhancing lesions. Due to the rarity of cerebral toxoplasmosis cases in Korea, the diagnosis and treatment are often delayed. This paper concerns a male patient whose cerebral toxoplasmosis was activated 21 years post kidney transplantation. Brain open biopsy was decided to make an exact diagnosis. Cerebral toxoplasmosis was confirmed by immunohistochemistry and PCR analyses of the tissue samples. Although cerebral toxoplasmosis was under control with medication, the patient did not recover clinically and died due to sepsis and recurrent gastrointestinal bleeding.     

Ocular impairment of toxoplasmosis

The purpose of this review is to update the latest information about ocular toxoplasmosis. The infection can be congenital or acquired, but also depends about the immune condition of the patient and can affect the eye. Ocular symptoms are variable according to the age of the subject. Retinochoroiditis is the most common manifestation of toxoplasmic infection. Toxoplasmic retinochoroiditis typically affects the posterior pole, and the lesions can be solitary or multiple. Active lesions present as grey-white focus of retinal necrosis with adjacent choroiditis, vasculitis, hemorrhage and vitreitis. Anterior uveitis is a common finding. Atypical presentations include punctate outer retinitis, neuroretinitis and papillitis. Depending on the patient's age and the localization of the lesion, ocular symptoms vary usually presenting with reduced visual acuity or without symptoms. The laboratory diagnosis of toxoplasmosis is based on detection of antibodies and T. gondii DNA using polymerase chain reaction (PCR) which fulfillis clinical findings. Toxoplasmosis therapy includes antimicrobial drugs and corticosteroids. There are several regimens with different drug combinations including, among others, pyrimethamine, sulfadiazine, clindamycin, and trimethoprim-sulfamethoxazol.     

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.     

Highly Active AntiRetroviral Therapy and opportunistic protozoan infections

Opportunistic parasite infections (OPIs) are an important cause of morbidity and mortality in persons infected with HIV. In industrialised countries, the use of Highly Active AntiRetroviral Therapy (HAART) results to be effective in suppressing the HIV viral load, with a quantitative and qualitative improvement in the CD4+ T-cell count followed by a strong reduction of opportunistic infections including those caused by parasites. These successes have been mainly attributed to the reconstitution of the cell immunity, which play the most important role in controlling OPIs. However, there are many clinical reports and several laboratory results, which suggest that the control of OPIs in HIV-positive persons under HAART is also induced by the anti-HIV protease inhibitors (PIs), which inhibit the aspartyl proteases of the parasites. The non-conventional use of HIV-PIs seems to be an alternative way for the treatment of parasitic infections, which should be deeply investigated. Of five longitudinal studies carried out before and after the introduction of HAART, four studies showed a strong reduction of toxoplasmic encephalitis (TE) in HIV-positive persons under HAART, whereas in another study, no difference was observed in the incidence rate of TE before and after the introduction of HAART. The influence of HAART in reducing TE has been also confirmed in a randomised, controlled clinical trial, which showed that there is no increase in the risk of developing TE after beginning HAART, even though HIV-infected persons with TE had a discontinuing prophylaxis for Toxoplasma gondii. Four HIV protease inhibitors were tested against the T. gondii virulent RH strain in vitro, alone or in association with pyrimethamine or sulfadiazine. Ritonavir and nelfinavir were highly inhibitory for the parasite growth. Furthermore, none of the antiviral drugs negatively affected the anti-Toxoplasma activity of pyrimethamine or sulfadiazine. In HIV-Leishmania co-infections, a changing pattern has been observed in the HAART era, characterised by a high rate of relapses, which could be explained by the increased survival rate resulting from the effective antiretroviral therapy. A 64.8% decrease of the visceral leishmaniasis incidence was detected after HAART began to be used extensively in Spain. In a large cohort study carried out in ten European countries and in Australia, the relative risk to contract cryptosporidiosis as the first AIDS defining disease was reduced by 96% in the HAART era. In Italy, the relative risk of death for cryptosporidiosis reduced of 74% in the period 1997-98, when HIV-positive persons received HAART. In a large study carried out in Italy, isosporiasis was included in the group of opportunistic infections, of which the relative hazards showed a reduction of 95% in the HAART era. Since 1997, there was the evidence that the use of HAART in persons with advanced HIV infection can improve chronic diarrhoea and lead to disappearance of Enterocytozoon bieneusi from the stools. Although the reconstitution of the cellular immunity seems to be the main factor influencing the reduction of OPIs in persons with AIDS who undergo HAART, there are clinical and microbiological evidences, as well as in vitro and in vivo results, which indicate direct effects of HIV-PIs on the proteases of opportunistic parasites. These findings stress the existence of non-conventional unexpected benefits of PIs in HAART against protozoa. In addition, this benefit of PIs has been demonstrated also for Candida albicans secreted aspartyl proteases and for P. carinii acid proteases. In spite of these important results, HIV PIs are still very toxic for humans, specially in cases of very long treatment, and no clinical trial has been carried out for persons at risk, such as children and pregnant women, because the priority was to reduce the severity of HIV and not the evaluation of possible side effects of the therapy. It follows that further researches are needed to establish the non-conventional use of HIV PIs. Furthermore, the study of PIs against specific aspartyl proteases of those opportunistic protozoa that cause severe and intractable diseases, could be considered an alternative way towards the development of new drugs that may prove effective against these infections.     

浆母细胞淋巴瘤的诊治进展

浆母细胞淋巴瘤是一种临床罕见、侵袭性极强的B细胞淋巴瘤,多发生在HIV感染的患者,也可见于HIV阴性的免疫功能正常或受抑的人群,常与EB病毒感染相关。由于其特殊的形态学及免疫表型特征、化疗耐药及复发率高的临床特点,诊断及治疗仍极具挑战性,总体预后极差。本文简要总结了PBL在流行病学、病理学特征、临床表现、诊断、治疗及预后因素等方面的进展,期望为PBL的临床诊治提供帮助。     

Is stage conversion the initiating event for reactivation of Toxoplasma gondii in brain tissue of AIDS patients?

Reactivation of chronic toxoplasmosis resulting in Toxoplasma encephalitis (TE) is a common event in acquired immune deficiency syndrome (AIDS) patients. Conversion from Toxoplasma gondii bradyzoites to tachyzoites is a prerequisite for reactivation. Until recently, the study of stage conversion in human tissue was not possible due to the lack of antibodies that recognize stage-specific epitopes after long-term formaldehyde fixation. Using the combination of a polyclonal anti-T. gondii antibody, the cyst-stage-specific monoclonal antibody CC2, and a tachyzoite-specific polyclonal antibody (anti-SAG1, recombinant), we tried to demonstrate parasite differentiation in the brain tissue of 10 AIDS patients with clinically suspected TE. Double labeling of the stage-specific antibodies enabled us to demonstrate interconversion between tachyzoites and bradyzoites for the first time in human tissue. The study confirmed that the transformation process is nonsynchronous and that the manifestation of TE depends on the degree and site of tissue destruction caused by invading tachyzoites. The original source of tachyzoites could never be located, but a few samples suggested that tachyzoites may invade by dissemination across the blood-brain barrier. Cyst rupture as the first event in the process of reactivation was not seen. We conclude that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical symptoms occur.     

Morbidity and mortality of red foxes (Vulpes vulpes) and gray foxes (Urocyon cinereoargenteus) admitted to the Wildlife Center of Virginia, 1993-2001

The medical records of 48 red foxes (Vulpes vulpes) and 35 gray foxes (Urocyon cinereoargenteus) examined at the Wildlife Center of Virginia (Waynesboro, Virginia, USA) from 1993 to 2001 were reviewed. The most common diagnosis in red foxes was orphaned (33%), followed by trauma (27%), undetermined diagnosis (23%), and sarcoptic mange (17%). Trauma (46%) was the most frequent cause of morbidity and mortality in gray foxes followed by orphaned (23%), undetermined (20%), toxoplasmosis (6%), presumptive canine distemper (3%), and rabies (3%). One gray fox had concurrent toxoplasmosis and presumptive canine distemper (3%). Similar diseases were detected in previous studies at a diagnostic laboratory; however in this study, trauma and orphaned animals were more common than infectious diseases. The lack of diagnostic information on some cases limited the usefulness of this study, and more emphasis should be placed on performing postmortem examinations of wildlife presented to wildlife rehabilitation centers.     

Western blotting for the diagnosis of congenital toxoplasmosis

Toxoplasmosis is a common congenital infection. It does not usually produce recognizable signs of infection at birth so most infected newborns are not detected by routine clinical examination and remain untreated. Infected children without clinical symptoms should nonetheless be identified and treated as early as possible. Serological diagnosis of congenital toxoplasmosis is quite difficult. The aim of this study was to evaluate the utility of Western blot for the diagnosis of congenital toxoplasmosis. We compared the immunological profiles of mothers and children to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants in the first three months of life. The method enabled us to diagnose congenital toxoplasmosis in cases in which the infection had not been detected by classical serology techniques.     

Seroprevalence of toxoplasmosis in Korean pregnant women

This study was performed in order to evaluate the sero-epidemiological status of toxoplasmosis in pregnant Korean women. Among 5,175 sera and 750 amniotic fluid samples obtained from pregnant women, 41 serum samples (0.79%) and 10 (1.33%) amniotic fluid samples tested positive for IgG antibodies by ELISA. Fifty one cases showing a score more than 0.25 on ELISA were tested for PCR reaction against the SAG1 gene. Only one case of the 51 ELISA positive cases exhibited a positive reaction on all tests. This case had a history of acute nephropyelitis during early pregnancy, but fortunately, had delivered a phenotypically healthy baby. In this study, the seroprevalence of toxoplasmosis in pregnant women was found to be comparatively low, consistent with previous reports from Korea. However our trials, performed with a variety of diagnostic tools, were considered to be useful for the precise diagnosis of congenital toxoplasmosis.     

Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections and studies on taxonomic classification

Lyme borreliosis caused by the spirochete Borrelia burgdorferi is now the most common vectorborne disease in North America, Europe and Asia. It is a multisystemic infection which may cause skin, neurological, cardiac or rheumatologic disorders. The aims of the present thesis were: (i) to develop a PCR assay for direct detection of B. burgdorferi DNA and to evaluate the diagnostic utility of PCR in clinical specimens from patients with Lyme borreliosis and (ii) to study the taxonomic classification of B. burgdorferi isolates and its implications for epidemiology and clinical presentation. Laboratory diagnosis of Lyme borreliosis by direct demonstration of B. burgdorferi in clinical specimens would compared to current serology allow (i) optimal specificity, (ii) increased sensitivity during the first weeks of infection, when the antibody response is not yet detectable and (iii) discrimination between ongoing and past infection. Due to the extreme paucity of spirochetes in clinical specimens neither in vitro culture nor antigen detection had yielded a sufficient diagnostic sensitivity. Thus the recently introduced highly sensitive PCR methodology could be a solution and was thus studied. Assays for PCR amplification and subsequent identification of B. burgdorferi specific sequences were established and used. For all assays the analytical sensitivity was a few genome copies using purified DNA as template. The efficacy of PCR was initially evaluated using tissue samples from experimentally infected gerbils in order to start with biological samples a priori known to contain B. burgdorferi. B. burgdorferi DNA was detectable in 88% of the specimens. Thus the diagnostic sensitivity of PCR was comparable to and even higher than in vitro culture. PCR was significantly more sensitive than a histological B. burgdorferi specific immunophosphatase-staining method. The utility of the PCR was then tested for identification of B. burgdorferi DNA in skin biopsies from 31 patients with erythema migrans. The sensitivity of PCR was 71%, which was superior to culture and serology. Based on own and otherwise published results there is clear evidence for PCR being the most sensitive and specific test for detection of B. burgdorferi in skin biopsies from patients with both early and late dermatoborreliosis. However, since the clinical diagnosis of dermatoborreliosis in most instances is easy, an invasive procedure as a skin biopsy, will only be justified in patients with an atypical clinical presentation. The most frequent and serious manifestation of disseminated Lyme borreliosis is neuroborreliosis. PCR was applied to 190 patients with untreated and confirmed neuroborreliosis. B. burgdorferi DNA was detectable in 17-21% of CSF samples from patients with neuroborreliosis. In patients with very early neuroborreliosis (< 2 weeks), still being negative for specific intrathecal antibody synthesis, a positive PCR was more frequent than in patients with longer disease duration. PCR can be used as a diagnostic aid in these patients. However, in general the measurement of specific intrathecal antibody production in patients with neuroborreliosis was superior to PCR. In urine samples from patients with Lyme borreliosis the diagnostic sensitivity varied, generally showing a low reproducibility. Urine is thus not regarded as a suitable sample source for B. burgdorferi PCR. The reason may be the variable presence of Taq polymerase inhibitors. Based on a semi-quantitative detection system for amplicons, reflecting the input amount of specific DNA and thus the density of spirochetes in the clinical samples high amounts of DNA were found in skin biopsies whereas especially in urine the amount of DNA was low. When the present study was initiated there was no accepted classification of B. burgdorferi. A heterogeneity among B. burgdorferi strains might have important implications for understanding the epidemiology and different clinical presentations (dermatoborreliosis versus neuroborreliosis) and courses (self-limiting versus chronic disease). Furthermore, strain differences were of importance for selection of suitable antigens for diagnostic assays and for vaccine development. Since then, B. burgdorferi isolates have been studied by phenotypic and genotypic traits and have been shown to be highly heterogeneous. Our first approach was to genotype a panel of human B. burgdorferi isolates by restriction fragment length polymorphism (RFLP) of three genes. Thereafter, sequencing and dideoxy fingerprinting of ospA was applied. By RFLP the strains could be differentiated into two to five groups. The RFLP classification was compared with four different phenotypic and genotypic methods including the rRNA typing. Results obtained with the different methods correlated highly and confirmed the meanwhile accepted taxonomic classification by Baranton et al., According to this the term B. burgdorferi sensu lato comprises three different human pathogenic genospecies B. burgdorferi sensu stricto, B. garinii and B. afzelii. All three genospecies have been isolated among Danish patients with Lyme borreliosis and are thus prevalent in Denmark. Since isolation of B. burgdorferi from patients with Lyme borreliosis is laborious and often unsuccessful molecular typing methods based on PCR are recommended obviating the need for isolation by prior culture. Of special interest was to study a possible association of neuroborreliosis to certain B. burgdorferi genospecies, indicating species depended organotropism. By RFLP all six CSF isolates tested belonged to B. garinii and that 6 out of 7 isolates from patients with acrodermatitis chronica atrophicans belonged to B. afzelii. Due to the low culture yield of B. burgdorferi from CSF, the association of B. garinii and neuroborreliosis was further studied by sequence analysis and dideoxyfingerprinting analysis of ospA PCR amplicons obtained from CSF samples from patients with neuroborreliosis. Phylogenetic analysis showed that in 11 out of 13 patients B. garinii DNA was found in CSF. These data strongly supports the hypothesis that B. garinii is the principal agent of Lyme neuroborreliosis in Europe. Similarly it was shown that B. afzelii is associated with acrodermatitis chronica atrophicans and thus dermatoborreliosis. Due to a strain dependent different selection pressure in culture only PCR based methods can be used to answer whether mixed infection in patients specimens occur. Our data indicate that mixed infections in humans if ever are rare.     

Determining Toxoplasma high-risk autologous and allogeneic hematopoietic stem cell transplantation patients by systematic pre-transplant PCR screening of stem cell originated buffy coat

The diagnosis of Toxoplasma infection or disease in hematopoietic stem cell transplantation (HSCT) patients is achieved mainly by PCR screening; however screening did not find wide field of use in practice due to costly expenditures of PCR. This study aimed to determine patients at high risk of Toxoplasma infection or disease before transplantation by stem cell originated buffy coat PCR and subsequently to screen them. Buffy coats collected from 12 autologous and 18 allogeneic HSCT patients' donors were investigated by PCR before transplantation. After transplantation, blood and sera collected at fixed time intervals were screened by two PCR methods and serological assays. Screening results first time assessed a toxoplasmosis incidence level as 25% in autologous HSCT patients and increased incidence level in allogeneic HSCT patients to 22%. Importantly, buffy coat PCR was first time performed before transplantation, to determine the risk of toxoplasmosis. Buffy coat PCR results showed that four patients were at high risk of toxoplasmosis before transplantation. After transplantation, these patients experienced toxoplasmosis. In conclusion, for the determination of patients at risk of toxoplasmosis, clinicians should consider buffy coat PCR in combination with serology before transplantation. After transplantation, PCR screening can be initiated in high risk patients upon clinical suspicion.     

Ischaemia with no obstructive coronary arteries

Ischaemia with no obstructive coronary arteries (INOCA) is a common ischaemic heart disease with a female preponderance, mostly due to underlying coronary vascular dysfunction comprising coronary microvascular dysfunction and/or epicardial coronary vasospasm. Since standard ischaemia detection tests and coronary angiograms are not suitable to diagnose coronary vascular dysfunction, INOCA is often overlooked in current cardiology practice. Future research, including large outcome trials, is much awaited. Yet, adequate diagnosis is possible and treatment options are available and vital to reduce symptoms and most probably improve cardiovascular prognosis. This review intends to give a brief overview of the clinical presentation, underlying pathophysiology, and the diagnostic and treatment options in patients with suspected INOCA.

     

An overview on the molecular diagnosis of animal leptospirosis

The most common presentation of animal leptospirosis is the subclinical and silent chronic form, that can lead to important reproductive disorders. The diagnosis of this chronic form remains a challenge. The aim of the present study is to gather and critically analyse the current information about molecular tools applied to animal leptospirosis diagnosis, particularly the silent chronic presentation of the infection. Regarding clinical specimens, samples from urinary tract were the most used (69/102, 67·7%), while few studies (12/102, 11·8%) investigated samples from reproductive tract. Concerning the molecular methods applied, the most used is still the conventional polymerase chain reaction (PCR) (46/102, 45%), followed by real-time PCR (38/102, 37·2%). The lipL32 gene is currently the most common target used for Leptospira detection, with 48% of studies applying this genetic marker. From all the studies, only few (21/102, 20·5%) performed gene sequencing. According to the majority of authors, current evidence suggests that lipL32-PCR is useful for an initial screening for Leptospira DNA detection in animal clinical samples. Posteriorly, if DNA sequencing could be performed on positive lipL32-PCR samples, we encourage the use of secY gene as a genetic marker. The molecular methods appear as the most important tools for the diagnosis of the chronic silent leptospirosis on domestic animals, reinforcing its evident impact not only on animal reproduction but also on a One Health context.     

Mitochondrial diseases in children including Leigh syndrome--biochemical and molecular background

Mitochondrial diseases in children are more frequently caused by mutations in nuclear DNA then in mtDNA. Special clinical phenotypes are associated with the mutations in SURF1 gene, in SCO2 gene and with mtDNA depletion syndromes. Leigh syndrome is the most common clinical presentation of various mitochondrial disorders during childhood. Elevation of lactate in blood, cerebrospinal fluid and urine is a simple biochemical marker of mitochondrial disorders but its specificity and sensitivity are low. Biochemical investigation of muscle biopsy and search for mitochondrial mutations remain a gold standard in the diagnosis. The standarized diagnostic criteria to establish level of diagnostic certainty (possible, probable, definite) are proposed to be used in practice; these include clinical features, neuroimaging and muscle biopsy investigations. Further research directions to improve our understanding of mitochondrial pathologies in children are suggested.     

Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.     

Diagnosis of tuberculous lymphadenitis by FNAC, microbiological methods and PCR: a comparative study

Despite its usefulness in the diagnosis of tuberculous lymphadenitis, fine needle aspiration cytology (FNAC) faces several limitations, and its sensitivity and specificity are not well established. The diagnostic accuracy and limitations of FNAC were studied in comparison with conventional microbiological methods and polymerase chain reaction (PCR). Sixty patients with lymphadenopathy and a clinical diagnosis of tuberculous lymphadenitis were subjected to FNA. The aspirate was used for cytological examination, Ziehl-Neelsen staining, mycobacterial culture and PCR. PCR was performed using two sets of oligonucleotide primers for Mycobacterium tuberculosis and a single primer for M. bovis species. The results of FNAC, microbiological methods and PCR correlated with the clinical outcome after follow-up for an average period of 24 months. Twenty-five cases (41.6%) were treated and responded well to anti-tuberculosis therapy, among them 17 were correctly diagnosed by FNAC (68%), eight by microbiological methods (32%) and 24 by PCR (96%). When PCR is considered the gold standard, FNAC predicted the correct diagnosis in 62% of cases with a high false negative rate (38%) due to the absence of granuloma/necrosis in smears from cases of early tuberculosis. In the latter group PCR proved to be the most valuable and a diagnostic success of 100% was achieved when FNAC and PCR were combined. In addition, PCR allowed immediate characterization of M. tuberculosis in the vast majority (96.2%) of cases in the study population.