Leflunomide and Polyomavirus-Associated Nephropathy in Renal Transplant

Polyomavirus nephropathy is a major complication in renal transplantation, associated with renal allograft loss in 14 to 80% of cases. There is no established treatment, although improvement has been reported with a variety of approaches. The authors report two cases of polyomavirus infection in renal allograft recipients. In the first case, a stable patient presented with deterioration of renal function, worsening hypertension and weight gain following removal of ureteral stent placed routinely at the time of surgery. Ultrasound examination and radiology studies revealed hydronephrosis due to ureteral stenosis. A new ureteral stent was placed, but renal function did not improve. Urinary cytology revealed the presence of decoy cells and polyomavirus was detected in blood and urine by qualitative polymerase chain reaction. Renal biopsy findings were consistent with polyomavirus -associated nephropathy. In the second case, leucopaenia was detected in an asymptomatic patient 6 months after transplantation. Mycophenolate mophetil dosage was reduced but renal allograft function deteriorated, and a kidney biopsy revealed polyomavirus -associated nephropathy, also with SV40 positive cells. In both patients immunosuppression with tacrolimus was reduced, mycophenolate mophetil stopped and intravenous immune globulin plus ciprofloxacin started. As renal function continued to deteriorate, therapy with leflunomide (40 mg/day) was associated and maintained during 5 and 3 months respectively. In the first patient, renal function stabilised within one month of starting leflunomide and polymerase chain reaction was negative for polyomavirus after 5 months. A repeated allograft biopsy 6 months later showed no evidence of polyomavirus nephropathy. In the second patient, polyomavirus was undetectable in blood and urine by polymerase chain reaction after 3 months of leflunomide treatment, with no evidence of polyomavirus infection in a repeated biopsy 6 months after beginning treatment.

Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

Adenovirus (AdV) respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF), and a specific nested polymerase chain reaction (PCR), to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%). In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.

Refractory pemphigus vulgaris associated with herpes infection: case report and review

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by blistering of the skin and mucosa, which develops due to the interaction between predisposing genetic and environmental factors. Infections caused by members of the Herpesviridae family have been suggested as a possible triggering factor for PV. OBJECTIVE AND METHODS: In this report, we investigate the presence of herpesviruses in refractory lesions on the right upper eyelid. The lesion has persisted despite the treatment with corticosteroids. Polymerase chain reaction (PCR) and DNA sequence analysis have been used to detect the DNA of HSV 1/2, VZV, EBV, CMV, HHV-6, HHV-7, and HHV-8. RESULTS: The sample collected from the right upper eyelid has tested positive for HSV 1/2. Sequence analysis has confirmed the PCR results and allowed the identification of the HSV strain as belonging to type 1. After treatment with acyclovir, the lesion of the right upper eyelid has cleared and not relapsed. CONCLUSION: When patients present PV lesions which are refractory to corticosteroid therapy, herpetic infection should be considered.

Evaluation of GBV-C / HVG viremia in HIV-infected women

The present study aimed at standardizing a real-time quantitative polymerase chain reaction assay to evaluate the presence of GBV-C/HGV RNA. A "TaqMan" assay using primers and probe derived from the 5¢ NCR region was developed and validated. Two hundred and fifty-three plasma samples from HIV-infected women were tested for GBV-C viremia and antibody against the envelope protein 2. GBV-C RNA was detected in 22.5% of the patients whereas the antibody was identified in 25.3% of the cohort. Detection of viral RNA and of antibodies was mutually exclusive. Viral loads showed a mean of 1,777 arbitrary units / mL, being 1.1 and 13,625 arbitrary units / mL respectively the lowest and highest values measured. We conclude that the real-time quantitative polymerase chain reaction method developed is appropriate for the investigation of GBV-C RNA since it was shown to be highly specific and sensitive, as well as requiring few steps, preventing contamination and providing additional information as to the relative viremia of carriers, a parameter that must be included in studies evaluating the co-factors influencing the clinical outcome of HIV/AIDS.

Diagnosing visceral leishmaniasis and HIV/AIDS co-infection: a case series study in Pernambuco, Brazil

HIV/AIDS-associated visceral leishmaniasis may display the characteristics of an aggressive disease or without specific symptoms at all, thus making diagnosis difficult. The present study describes the results of diagnostic tests applied to a series of suspected VL cases in HIV-infected/AIDS patients admitted in referral hospitals in Pernambuco, Brazil. From a total of 14 eligible patients with cytopenias and/or fever of an unknown etiology, and indication of bone marrow aspirate, 10 patients were selected for inclusion in the study. Diagnosis was confirmed by the following examinations: Leishmania detection in bone marrow aspirate, direct agglutination test, indirect immunofluorescence, rK39 dipstick test, polymerase chain reaction and latex agglutination test. Five out of the ten patients were diagnosed with co-infection. A positive direct agglutination test was recorded for all five co-infected patients, the Leishmania detection and latex agglutination tests were positive in four patients, the rK39 dipstick test in three, the indirect immunofluorescence in two and a positive polymerase chain reaction was recorded for one patient. This series of cases was the first to be conducted in Brazil using this set of tests in order to detect co-infection. However, no consensus has thus far been reached regarding the most appropriate examination for the screening and monitoring of this group of patients.

Expression of TLR2 and TLR4 in lesions of patients with tegumentary american leishmaniasis

OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-α, anti-IFN-Γ, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 ± 82.46 cell/mm² and 3.26 ± 4.11 cell/mm² respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.

Whipple's disease: rare disorder and late diagnosis

Whipple's disease is a rare systemic infectious disorder caused by the bacterium Tropheryma whipplei. We report the case of a 61-year-old male patient who presented to emergency room complaining of asthenia, arthralgia, anorexia, articular complaints intermittent diarrhea, and a 10-kg weight loss in one year. Laboratory tests showed the following results: Hb = 7.5 g/dL, albumin = 2.5 mg/dL, weight = 50.3 kg (BMI 17.4 kg/m²). Upper gastrointestinal endoscopy revealed areas of focal enanthema in the duodenum. An endoscopic biopsy was suggestive of Whipple's disease. Diagnosis was confirmed based on a positive serum polymerase chain reaction. Treatment was initiated with intravenous ceftriaxone followed by oral trimethoprim-sulfamethoxazole. After one year of treatment, the patient was asymptomatic, with Hb = 13.5 g/dL, serum albumin = 5.3 mg/dL, and weight = 70 kg (BMI 24.2 kg/m²). Whipple's disease should be considered a differential diagnosis in patients with prolonged constitutional and/or gastrointestinal symptoms. Appropriate antibiotic treatment improves the quality of life of patients.

TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

NATURAL INFECTION BY Trypanosoma cruzi IN ONE DOG IN CENTRAL WESTERN BRAZIL: A CASE REPORT

SUMMARY It is estimated that about 10 million people are infected with Trypanosoma cruzi worldwide, mostly in Latin America and more than 25 million are at risk of acquiring this infection in endemic areas. Dogs are an important reservoir for this pathogen and thus, considered a risk factor for human populations. This report describes one case of Chagas disease in a dog from Cuiabá, Mato Grosso State, Brazil. The diagnosis was obtained by direct examination of trypomastigote forms in blood smears. Amastigotes forms were visualized in microscopy of the bone marrow, lymph nodes, kidneys, liver and brain. The T. cruzi (ZIII) infection was confirmed by Polymerase Chain Reaction, and sequencing. The animal presented multisystemic failure and died. Although acute Chagas disease in humans is not reported in Cuiabá, this is the first report of a canine case in this region. This case represents a warning, to health professionals and authorities, to the possibility of transmission of this zoonosis in Cuiabá.

PREVALENCE OF HERPES SIMPLEX VIRUS TYPE 2 AND RISK FACTORS ASSOCIATED WITH THIS INFECTION IN WOMEN IN SOUTHERN BRAZIL

SUMMARY The herpes simplex virus type 2 (HVS-2) is the most prevalent infection worldwide. It is a cofactor in the acquisition of human immunodeficiency virus (HIV) and the persistence of human papillomavirus (HPV). This study evaluated the prevalence of HSV-2, using the polymerase chain reaction (PCR), and associated factors in patients treated at the Federal University of Rio Grande (FURG) and Basic Health Units (BHU) in Rio Grande, Brazil. The observed prevalence of HSV-2 was 15.6%. Among the 302 women studied, 158 had received assistance in BHU and 144 were treated at FURG. The prevalence of HSV-2 in these groups was 10.8% and 20.8%, respectively, RR 1.9 and p = 0.012. Knowledge about the Pap smear, and the presence of lesions showed no association with HSV-2 infection. Multivariate analysis showed that the variable that most influenced the risk of HSV-2 infection was the presence of HIV infection, with a relative risk of 1.9 and p = 0.04. Discussion: Genital ulcers are an important entry point for HIV, and condom use is an important strategy to reduce transmission of HIV and HSV-2.

FIRST REPORT ON Cryptococcus neoformans IN PIGEON EXCRETA FROM PUBLIC AND RESIDENTIAL LOCATIONS IN THE METROPOLITAN AREA OF CUIABÁ, STATE OF MATO GROSSO, BRAZIL

SUMMARYCryptococcosis is a severe systemic mycosis caused by two species of Cryptococcus that affect humans and animals: C. neoformans and C. gattii. Cosmopolitan and emergent, the mycosis results from the interaction between a susceptible host and the environment. The occurrence of C. neoformanswas evaluated in 122 samples of dried pigeon excreta collected in 49 locations in the City of Cuiabá, State of Mato Grosso, Brazil, including public squares (n = 5), churches (n = 4), educational institutions (n = 3), health units (n = 8), open areas covered with asbestos (n = 4), residences (n = 23), factory (n = 1) and a prison (n = 1). Samples collected from July to December of 2010 were seeded on Niger seed agar (NSA). Dark brown colonies were identified by urease test, carbon source assimilation tests and canavanine-glycine-bromothymol blue medium. Polymerase chain reaction primer pairs specific for C. neoformans were also used for identification. Cryptococcus neoformans associated to pigeon excreta was isolated from eight (6.6%) samples corresponding to six (12.2%) locations.Cryptococcus neoformans was isolated from urban areas, predominantly in residences, constituting a risk of acquiring the disease by immunocompromised and immunocompetent individuals.

USEFULNESS OF kDNA PCR IN THE DIAGNOSIS OF VISCERAL LEISHMANIASIS REACTIVATION IN CO-INFECTED PATIENTS

SUMMARYIt is important to develop new methods for diagnosing relapses in the co-infection of visceral leishmaniasis (VL) and HIV to enable earlier detection using less invasive methods. We report a case of a co-infected patient who had relapses after VL treatment, where the qualitative kDNA PCR showed a good performance. The kDNA PCR seems to be a useful tool for diagnosing VL and may be a good marker for predicting VL relapses after treatment of co-infected patients with clinical symptoms of the disease.

QUANTITATIVE REAL-TIME PCR (Q-PCR) FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

Objective: To assess quantitative real-time polymerase chain reaction (q-PCR) for the sputum smear diagnosis of pulmonary tuberculosis (PTB) in patients living with HIV/AIDS with a clinical suspicion of PTB.Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard.Results: Of the 140 sputum samples, 47 (33.6%) were positive with the gold standard. q-PCR was positive in 42 (30%) of the 140 patients. Only one (0.71%) did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93%) of the 42 q-PCR positive cases, the CT (threshold cycle) was equal to or less than 37.Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.

MOLECULAR IDENTIFICATION OF Bartonella henselae IN A SERONEGATIVE CAT SCRATCH DISEASE PATIENT WITH AIDS IN RIO DE JANEIRO, BRAZIL

Bartonella henselae is associated with a wide spectrum of clinical manifestations, including cat scratch disease, endocarditis and meningoencephalitis, in immunocompetent and immunocompromised patients. We report the first molecularly confirmed case of B. henselae infection in an AIDS patient in state of Rio de Janeiro, Brazil. Although DNA sequence of B. henselae has been detected by polymerase chain reaction in a lymph node biopsy, acute and convalescent sera were nonreactive.