Christian Vandenvelde

Fast Multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis

An assessment of optimal conditions for rapid simultaneous amplification of multiple human papillomavirus (HPV) sequences has been made using Thermus aquaticus DNA polymerase. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of well characterized cell lines. In our hands, Fast Multiplex PCR (FM-PCR), the technique of running multiple PCR reactions simultaneously with minimum incubation time at each temperature, was highly sensitive (amplification factor = 5 x 10(9) after 50 cycles), specific (100%) and reproducible (100%) for several microbiological applications. Diagnosis was generally obtained in less than 5 h after sampling. The results show that, after optimization of assay conditions, efficiency and specificity of Multiplex PCR depends exclusively on the primers design and concentration of the primers.

Human anaplasmosis in Belgium: A 10-year seroepidemiological study

Human granulocytic anaplasmosis (HGA) is a tick-borne rickettsial infection of neutrophils caused by Anaplasma phagocytophilum. Although the pathogen was known as a veterinary agent as early as 1932, the link with human disease was first established in 1990. In the past decennium, the involvement of HGA as an important and frequent cause of fever with a history of tick bite was increasingly recognized in many regions of Europe. This paper presents a 10-year A. phagocytophilum serosurveillance (2000-2009), wherein 1672 serum samples were tested and 418 were found positive. A total of 111 patients had a history of tick bite, fever, and at least a 4-fold rise in titre and are thus considered to be confirmed cases. These findings suggest that Belgium is a hot spot for HGA infections.

Distribution of hantavirus foci in Belgium

During 1999 and 2000, we performed rodent captures on 15 sites all over Belgium to evaluate the presence of hantaviruses in local rodent populations. Viral antibody and RNA detection was performed by ELISA/focus reduction neutralisation test and RT-PCR, respectively. We found hantavirus-positive rodents on 13 out of 15 trapping sites and 3 rodent species were found positive for hantavirus infection. Apart from Puumala virus that was carried by Clethrionomys glareolus, 2 additional rodent species, Microtus arvalis and Apodemus sylvaticus, were found antibody- and/or RNA-positive.

Association between habitat and prevalence of hantavirus infections in bank voles (Myodes glareolus) and wood mice (apodemus sylvaticus)

In order to determine the habitat preferred by Myodes (before Clethrionomys) glareolus and the corresponding Puumala hantavirus seroprevalence in those habitats, we captured rodents simultaneously in three significantly different habitats. We compared trapping success and presence of virus per habitat during an ongoing epidemic in order to test the hypothesis of a density-dependent seroprevalence. Our study showed that bank vole population density, as well as Puumala virus seroprevalence, were habitat dependent. Apodemus sylvaticus was found more vulnerable for deteriorating habitat conditions than M. glareolus and could play a role as vehicle for Puumala virus and as mediator for inter- and conspecific virus transmission.

Suppression of the inhibitory effect of denatured albumin on the polymerase chain reaction by sodium octanoate : application to routine clinical detection of hepatitis B virus at its infectivity threshold in serum

Ten to fifteen percent of posttransfusion viral hepatitis cases are still caused by HBV despite mandatory third generation screening procedures for HBsAg. There is thus an urgent need for a simple, time-cost-effective, but very sensitive test for routine HBV DNA detection in serum. Nested-primed PCR has been shown to detect purified HBV DNA at its infectivity threshold in serum. Since this is too labor-intensive for routine testing, we assessed the efficiency of a Fast PCR procedure, of three pairs of primers, and of thirty-five simple serum pretreatments with the aim to achieve the same sensitivity level. Using ten-fold dilution in phosphate buffered saline as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at the 2 x 10(3)/ml level in serum. Using either NaOH denaturation or sodium octanoate thermoprotection as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at its infectivity threshold in serum, while the classical phenol/chloroform/isoamylic alcohol/isopropanol/ethanol DNA purification procedure enabled us to reach the 10 virus particles/ml level. These results suggest that denatured albumin is responsible for the well known inhibitory effect of serum proteins on Taq polymerase. Because of its simplicity and its lower risk of sample-to-sample cross-contamination, the sodium octanoate thermoprotection method was chosen for routine clinical detection of HBV in serum. The clinical usefulness of this approach is demonstrated by the results obtained with HBsAg-negative acute hepatitis B incubation sera and with anti HBe-positive chronic hepatitis B sera.

Evaluation of a commercial enzyme immunomembrane filter assay for detection of respiratory syncytial virus in clinical specimens.

A commercial enzyme immunomembrane filter assay (EIFA) for respiratory syncytial virus (RSV) was compared prospectively with isolation in cell culture and an enzyme immunoassay. A total of 595 respiratory specimens, mostly from pediatric patients, were examined. The EIFA was 70.96 % sensitive and 72.40 % specific in comparison with cell culture. Results for 40 specimens (6.72 %) were uninterpretable, mainly due to filtration difficulties. Twenty-one (25 %) of 84 specimens whose results were initially considered false-positive were subsequently confirmed positive after a blocking test with bovine anti-RSV serum. On the basis of the total number of confirmed positive results, the sensitivity and the specificity of the test were 87.90 % and 75.77 %, respectively.

Potassium content of irradiated packed red blood cells in different storage media: Is there a need for additive solution-dependent recommendations for infant transfusion?

Prevention of transfusion-associated graft versus host disease (TA-GVHD) by gamma irradiation is known to induce increased K+ in supernatant of packed red blood cells (PRBCs) stored in CPDA-1 and SAGM conservative solutions. However, no data exist for PRBCs in AS-3 medium which is considered safe for neonatal transfusion. We evaluated haemolysis and K+ release from irradiated AS-3 PRBCs and compared our results with reported data for SAGM and CPDA-1 PRBCs. Our results indicate that irradiated PRBCs stored in AS-3 after more than 7 days post-irradiation should not be used in massive and/or rapidly infused transfusions in neonates and infants.

Fast Multiplex Polymerase Chain Reaction on Boiled Clinical Samples for Rapid Diagnosis of Viral Infections

In spite of the recent dramatic development of new, rapid methods for detection of viral infections, most laboratory diagnosis of viral disease still depends either on the growth of virus or on the detection of a specific serologic reaction to infection. As tissue culture is the most widely sensitive and flexible, it remains the golden standard. But, because no single monolayer type is sensitive to the growth of all viruses most laboratories choose an array of different tissue cultures and, depending on the suspected viruses, inoculate each specimen into those lines or strains most likely to give a positive result. For the most part each virus, or virus group, produces a characteristic cytopathic effect, and the skilled technologist can usually develop a tentative identification on the basis of cell tropism and morphology, giving the clinician useful information at the earliest possible moment. Final identification of virus isolates usually depends on serologic tests and sometimes on morphologic, biophysical, or biologic characterization. The polymerase chain reaction (PCR) has the potential to replace these laborious viral isolation methods, but problems such as specificity, reproducibility, rapidity, cost, and the need for a common sequence in all strains of a particular virus must be taken into account. The production of low and high molecular weight non-specific PCR products must be noted while using most published PCR protocols. The accumulation of such PCR-products depletes primers and deoxynucleotide-triphosphates from the reaction mixture and leads to competition for enzyme with the desired PCR-product. Diagnosis must then rely on final detection assays such as hybridization with radioactive oligonucleotide probes to guarantee sufficient specificity, sensitivity and reproducibility.