COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

INVESTIGATION OF BIOFILM FORMATION IN COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATED FROM PLATELET CONCENTRATE BAGS

Platelet Concentrates (PCs) are the blood components with the highest rate of bacterial contamination, and coagulase-negative staphylococci (CoNS) are the most frequently isolated contaminants. This study investigated the biofilm formation of 16 contaminated units out of 691 PCs tested by phenotypic and genotypic methods. Adhesion in Borosilicate Tube (ABT) and Congo Red Agar (CRA) tests were used to assess the presence of biofilm. The presence of icaADC genes was assessed by means of the Polymerase Chain Reaction (PCR) technique. With Vitek(r)2, Staphylococcus haemolyticus was considered the most prevalent CoNS (31.25%). The CRA characterized 43.8% as probable biofilm producers, and for the ABT test, 37.5%. The icaADC genes were identified in seven samples by the PCR. The ABT technique showed 85.7% sensitivity and 100% specificity when compared to the reference method (PCR), and presented strong agreement (k = 0.8). This study shows that species identified as PCs contaminants are considered inhabitants of the normal skin flora and they might become important pathogens. The results also lead to the recommendation of ABT use in laboratory routine for detecting biofilm in CoNS contaminants of PCs.

Sensitivity of Gomez-Nuñez boxes for the detection of household infestation with Panstrongylus megistus

Four methods for detecting household infestations with Panstrongylus megistus were compared: 1) manual collection; 2) collection after pyrethrum application; 3) search viable eggs; and 4) Gomez-Nuñez boxes. Manual collection was the most sensitive method (23% infested), followed by pyrethrum (21%), Gomez-Nuñez boxes (15%) and viable eggs (12%). About 10% of infested houses were positive exclusively on the Gomez-Nuñez box test. More over, 6 out of the 7 houses positive exclusively on the Gomez-Nuñez method were located in a recently sprayed area, where P. megistus density was low. Inspection of Gomez-Nuñez boxes at 12 weekspost-application was twice as effective as inspection at 6 weekspost-application. Triatomine feces was the most common evidencefor thepresence of P. megistus found within Gomez-Nuñez boxes. Gomez-Nuñez boxes area a useful adjunct to manual collection in detecting domestic infestations with P. megistus, especially in areas where bug densities are low. However, the utility of Gomez-Nuhez boxes must be weighed against the time and labor they require.

Parasitological diagnosis of mucocutaneous leishmaniasis due to Leishmania b. braziliensis in Bolivia

Parasitological diagnosis, using staned smears, culture and pathological examination of biopsy, was studied in 146 patients infected with mucocutaneous leishmaniasis, in Bolivia and Peru. The most efficient parasite detecting technique appeared to be the smear examination in cutaneous lesions (33 % positive) and the pathology in case of mucous lesions (28 % positive). In both, cutaneous and mucous lesions, the parasites were found most frequently in old lesions.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in Northern Brazil

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

Entamoeba histolytica and Entamoeba dispar infections as detected by monoclonal antibody in an urban slum in Fortaleza, Northeastern Brazil

In this study the authors used the Elisa-based antigen detection tests that distinguish E. histolytica from E. dispar to examine the prevalence of E. histolytica infection in individuals from an urban slum in Fortaleza, Northeastern, Brazil. This test has a sensitivity and specificity that is comparable to PCR and isoenzyme analysis, which is the gold standard. Single stools samples were obtained from 735 individuals. The prevalence of E. histolytica infection was 14.9% (110/735) and 25.4%(187/735) for E. dispar-E. histolytica complex. The most affected age group for E. histolytica /E. histolytica-E. dispar infection was the 1-5 year olds but there was no remarkable decrease with age. There was no significant difference in colonization rates between males and females. The results from this survey demonstrate that E. histolytica is highly prevalent in the Community studied. Furthermore, it offers promise for the antigen detection test as a sensitive and technically simple tool for detecting E. histolytica infection in the field.

Evaluation of Taenia solium and Taenia crassiceps cysticercal antigens for immunodiagnosis of neurocysticercosis using ELISA on cerebrospinal fluid samples

The efficacy of whole parasite and vesicular fluid antigen extracts from Taenia solium and Taenia crassiceps cysticerci for immunodiagnosis of neurocysticercosis was evaluated using ELISA on cerebrospinal fluid samples. Anticysticercal IgG antibodies were assayed in cerebrospinal fluid samples from 23 patients with neurocysticercosis and 35 patients with other neurological disorders. The ELISA reaction for the whole Taenia solium cysticercal extract showed 91.3% sensitivity and 94.3% specificity, whereas the sensitivity and specificity of the ELISA for the whole Taenia crassiceps cysticercal extract were 87% and 94.3%, respectively. The ELISA reactions for vesicular fluid from Taenia solium or Taenia crassiceps showed 91.3% sensitivity and 97.1% specificity. Considering the results obtained from the four antigen preparations, vesicular fluid from Taenia solium and Taenia crassiceps cysticerci may be useful as a source of antigens for immunological reactions that are used for detecting specific antibodies in cerebrospinal fluid samples from patients with neurocysticercosis.

Clinical and ultrasound findings before and after praziquantel treatment among Venezuelan schistosomiasis patients

Abdominal ultrasound can be a useful tool for diagnosing periportal fibrosis related to Schistosoma mansoni infection, and also for planning and monitoring the evolution of hepatic morbidity following control measures. We evaluated the standardized ultrasound methodology proposed by the World Health Organization for detecting periportal fibrosis and portal hypertension, among patients from an endemic area in Venezuela, and the impact of praziquantel treatment 3-5 years later. After chemotherapy, complete reversal of periportal lesions was observed in 28.2% of the cases and progression of the disease in 5.1%. Improvement in the hepatic disease started with a reduction in the periportal thickening followed by a decrease in the size of the left hepatic lobe, spleen and mesenteric and spleen veins. Ultrasound confirmed the clinical findings after chemotherapy among the patients with reversal of the disease. However, in patients with more advanced disease, these findings were contradictory. There was no correlation between evolution of the disease seen on ultrasound and age, intensity of infection or serological findings.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

Gastroenteric virus detection in fecal samples from women in Goiânia, State of Goiás, Brazil

INTRODUCTION: This was a prospective study that included women seen in the obstetrics and gynecology sector of Hospital das Clínicas, Federal University of Goiás, in Goiânia, State of Goiás, with the aim of detecting rotaviruses, adenoviruses, caliciviruses and astroviruses. Eighty-four women participated in the study and from these, 314 fecal samples were collected. Out of all of the women, 29 were seropositive for HIV and 55 were seronegative, and 45 and 39 were pregnant and non-pregnant, respectively. METHODS: Fecal samples were collected from each woman once every two months over the period from July 2006 to June 2007, and they were screened for rotaviruses by means of polyacrylamide gel electrophoresis and immunoenzymatic assays, for caliciviruses and astroviruses by means of RT-PCR and for adenovirus by means of immunoenzymatic assays. The astroviruses were genotyped using nested PCR. RESULTS: Among the 84 patients, 19 (22.6%) were positive for either calicivirus (14/19) or astrovirus (6/19), while one women was positive for both viruses in fecal samples collected on different occasions. Most of the positive samples were collected during the months of July and August (astrovirus) and September and October (calicivirus). None of the samples analyzed was positive for rotavirus or adenovirus. Gastroenteric viruses were detected in 13/19 (68.4%) of the pregnant women, whether HIV-seropositive or not. CONCLUSIONS: The results from the present study showed that neither pregnancy nor HIV-seropositive status among the women increased the risk of infection by any of the gastroenteric viruses studied. This study presents data on gastroenteric virus detection among pregnant and/or HIV-positive women.

Cocirculation of two dengue virus serotypes in individual and pooled samples of Aedes aegypti and Aedes albopictus larvae

INTRODUCTION: To detect dengue virus, eggs of Aedes sp were collected in the city of Belo Horizonte, Brazil, in 2007. METHODS: Egg samples were subsequently hatched and the larvae were tested for the presence of dengue virus RNA by RT-PCR. RESULTS: Among the Aedes aegypti larvae samples, 163 (37.4%) out of 435 were positive, including 32 (10.9%) of 293 individual larvae samples concomitantly positive for two serotypes. CONCLUSIONS: Virological surveillance detecting coinfected vectors in the field could represent an important strategy for understanding the numerous factors involved in the transmission and clinical presentation of dengue.

Hepatitis B and C virus infection among Brazilian Amazon riparians

INTRODUCTION: Viral hepatitis is a major public health concern in Brazil. There are few past studies on this issue, especially among riparian communities. This study aims at determining the seroprevalence of viral hepatitis B and C in the riparian community of Pacuí Island, within the Cametá municipality of Pará State, Brazil. Moreover, this study aims to investigate the principal risk factors that this community is exposed to. METHODS: The current study has accessed blood samples from 181 volunteers who have answered an epidemiological questionnaire. Analyses on serological markers have been tested with commercial ELISA kits for detecting HBsAg, total anti-HBc, anti-HBs, and anti-HCV. Within seroreactive patients for HCV, RT-PCR and line probe assay have been performed to identify the viral genotype. RESULTS: In the serological marker analysis for hepatitis B, no reactivity for HBsAg, rate of 1.1% for total anti-HBc, and rate of 19.3% for anti-HBs have been observed. On hepatitis C, 8.8% seroprevalence has been found, in which 62.5% have gotten viral RNA. Among the risk factors studied, the following have been highlighted: non-use of condoms, sharing of cutting instruments, use of illicit drugs, and reports of family disease with HBV or HCV. CONCLUSIONS: The vaccination coverage against HBV is low, and the high prevalence of HCV within this community has been observed.

Acute schistosomiasis diagnosis: a new tool for the diagnosis of schistosomiasis in a group of travelers recently infected in a new focus of Schistosoma mansoni

IntroductionThe diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group.MethodsWe describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings.ResultsELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg.ConclusionsOur data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.