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.

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.

FATAL OUTCOME OF INFECTION BY DENGUE 4 IN A PATIENT WITH THROMBOCYTOPENIC PURPURA AS A COMORBID CONDITION IN BRAZIL

Dengue is currently a major public-health problem. Dengue virus (DENV) is classified into four distinct serotypes, DENV 1-4. After 28 years of absence, DENV-4 was again detected in Brazil in 2010 in Roraima State, and one year later, the virus was identified in the northern Brazilian states of Amazonas and Pará, followed by Rio de Janeiro and São Paulo. In Minas Gerais, the first confirmed case of DENV-4 occurred in the municipality of Frutal in 2011 and has now been isolated from a growing number of patients. Although DENV-2 is associated with the highest risk of severe forms of the disease and death due to the infection, DENV-4 has also been associated with severe forms of the disease and an increasing risk of hemorrhagic manifestations. Herein, the first fatal case of confirmed DENV-4 in Brazil is reported. The patient was an 11-year-old girl from the municipality of Montes Claros in northern Minas Gerais State, Brazil. She had idiopathic thrombocytopenic purpura as a comorbid condition and presented with a fulminant course of infection, leading to death due to hemorrhagic complications. Diagnosis was confirmed by detection of Dengue-specific antibodies using IgM capture enzyme-linked immunosorbent assay and semi-nested RT-PCR. Primary care physicians and other health-care providers should bear in mind that DENV-4 can also result in severe forms of the disease and lead to hemorrhagic complications and death, mainly when dengue infection is associated with coexisting conditions.

PARTICIPATION OF TICKS IN THE INFECTIOUS CYCLE OF CANINE VISCERAL LEISHMANIASIS, IN TERESINA, PIAUÍ, BRAZIL

In this study, we detected Leishmania spp. infection in R. sanguineus collected from dogs that were naturally infected with L. (L.) infantum. We examined 35 dogs of both sexes and unknown ages. The infected dogs were serologically positive by the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Quick Test-DPP (Dual Path Platform), as well as parasitological examination of a positive skin biopsy or sternal bone marrow aspiration. Ten negative dogs were included as controls. The ticks that infested these dogs were collected in pools of 10 adult females per animal. The PCR was performed with specific primers for Leishmania spp., which amplified a 720-bp fragment. Of the 35 analyzed samples, a product was observed in eight samples (8/35; 22.9%). We conclude that the presence of parasite DNA suggests that ticks participate in the zoonotic cycle of canine visceral leishmaniasis, in the city of Teresina, Piauí.

FALSE-NEGATIVE DENGUE CASES IN RORAIMA, BRAZIL: AN APPROACH REGARDING THE HIGH NUMBER OF NEGATIVE RESULTS BY NS1 AG KITS

Serum samples from 150 NS1-negative (Platelia ELISA) patients presumptively diagnosed with dengue were analyzed by the TaqMan probed real-time reverse transcription PCR (TaqMan qRT-PCR) method. The qRT-PCR positive samples were tested for serotype by semi-nested RT-PCR and a qualitative immunochromatographic assay for IgG and IgM. Molecular detection methods showed 33 (22%) positive samples out of 150 NS1-antigen negative samples. Of these, 72% were collected up to day 2 after the onset of symptoms, when diagnostic sensitivity of NS1-antigen test assays is significantly enhanced. Most of the cases were not characterized as secondary infection. Twenty-eight samples were successfully serotyped, 75% of which for DENV-4, 14% for DENV-2, 7% for DENV-3 and 4% for DENV-1. These findings reaffirm the hyperendemic situation of the state of Roraima and suggest a lower sensitivity of the NS1 test, mainly when DENV-4 is the predominant serotype. Health care providers should therefore be aware of samples tested negative by NS1 antigen assays, especially when clinical symptoms and other laboratory data results show evidence of dengue infection.

QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES

SUMMARY Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

SUMMARYAIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex) or enzyme-linked immunoassay (EIA) has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA) was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered). CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcusspecies. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die.

Diagnostic tests for amoebic liver abscess: comparison of enzyme - linked immunosorbent assay (Elisa) and counterimmunoelectrophoresis (CIE)

The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoeletrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results iin both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100%) than that of the CIE technique (66%).

Detection of HTLV-IIa in blood donors in an urban area of the Amazon Region of Brazil (Belém, PA)

The human lymphotropic viruses type I (HTLV-I) and type II (HTLV-II) are members of a group of mammalian retroviruses with similar biological properties, and blood transfusion is an important route of transmission. HTLV-I is endemic in a number of different geographical areas and is associated with several clinical disorders. HTLV-II is endemic in several Indian groups of the Americas and intravenous drug abusers in North and South America, Europe and Southeast Asia. During the year of 1995, all blood donors tested positive to HTLV-I/II in the State Blood Bank (HEMOPA), were directed to a physician and to the Virus Laboratory at the Universidade Federal do Pará for counselling and laboratory diagnosis confirmation. Thirty-five sera were tested by an enzyme immune assay, and a Western blot that discriminates HTLV-I and HTLV-II infection. Two HTLV-II positive samples were submitted to PCR analysis of pX and env genomic region, and confirmed to be of subtype IIa. This is the first detection in Belém of the presence of HTLV-IIa infection among blood donors. This result emphasizes that HTLV-II is also present in urban areas of the Amazon region of Brazil and highlights the need to include screening tests that are capable to detect antibodies for both types of HTLV.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.