Morphological alterations of hippocampal pyramidal neurons heterotopically transplanted into the somatosensory cortex of adult rats: a quantitative Golgi study

A characteristic feature of hippocampal organization is the laminated termination of extrinsic and intrinsic afferents. At present, it is not known to what extent these layer-specific fiber projections modulate the development and final shape of the dendritic arbor of hippocampal target neurons. In the present study, pieces of late embryonic (E18) rat hippocampus were transplanted heterotopically into a cavity in the somatosensory cortex of 6–8 week-old recipient rats. Here, the transplanted neurons differentiated and survived up to several months in the absence of their specific extrinsic afferents. Moreover, tracing of transplant connections with the carbocyanine dye DiI revealed only a limited projection between the transplant and the host neocortex. Golgi-impregnated transplants were used to analyze the postsynaptic structures (dendrites and spines) of hippocampal pyramidal cells quantitatively. Compared with controls, the transplanted pyramidal neurons showed a significant reduction of apical primary dendrites and basal dendritic branches, i.e. of peripheral dendritic portions that originate farther from the soma. In contrast, the number of basal primary dendrites originating directly from the perikaryon was enhanced. Spine density on the main apical dendritic shaft was significantly lower in all peripheral dendritic segments in transplanted neurons. We conclude from our results that the absence of layerspecific extrinsic afferents that normally terminate on peripheral parts of the dendritic arbor of hippocampal pyramidal neurons caused a reduction of these peripheral dendrites and spines. In contrast, the increase of dendrites and spines near the cell body might be induced by intrinsic fibers that normally terminate on these proximal dendritic portions and are known to sprout under transplant conditions.     

The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation

Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N = 109) and negative (N = 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55–76) and 59% (95% CI: 49–68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N = 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used.     

Limited diagnostic capacities of two commercial assays for the detection of Leptospira immunoglobulin M antibodies in Laos

The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using admission serum (Leptotek sensitivity of 47.3% and specificity of 75.5%: ELISA sensitivity of 60.9% and specificity of 65.6%) compared to the Leptospira "gold standard" microscopic agglutination test.     

Temperature and the Field Stability of a Dengue Rapid Diagnostic Test in the Tropics

The global incidence of dengue has increased significantly in recent decades, resulting in a large public health burden in tropical and subtropical countries. Dengue rapid diagnostic tests (RDTs) can provide accurate, rapid accessible diagnosis for patient management and may be easily used by health workers in rural areas. However, in dengue-endemic areas, ambient temperatures are often higher than manufacturer''s recommendation. We therefore evaluated the effect of high temperature over time on the performance of one commonly used dengue RDT, the Standard Diagnostics Bioline Dengue Duo. RDTs were kept in five different conditions (at 4°C, 35°C, 45°C, 60°C, and at fluctuant ambient temperatures in a free-standing hut) for between 2 days and 2 years in the Lao People''s Democratic Republic (PDR). RDTs were tested with four control sera (negative, dengue nonstructural protein 1 [NS1], anti-dengue immunoglobulin [Ig] M, and anti-dengue IgG positive). The RDTs had 100% consistency over the 2-year study, despite high temperatures, including in the hut in which temperatures exceeded the manufacturer''s recommendations for 29% of time points. These data suggest that the diagnostic accuracy of the SD Bioline Dengue Duo RDT remains stable even after long-term storage at high temperatures. Therefore, use at such ambient temperatures in tropical areas should not jeopardize the dengue diagnostic outcome.     

Evaluation of the Standard Diagnostics Leptospira IgM ELISA for diagnosis of acute leptospirosis in Lao PDR

The diagnostic utility of the Standard Diagnostics Leptospira IgM ELISA for detection of acute leptospirosis was assessed in febrile adults admitted in Vientiane, Laos. Using the cut-off suggested by the manufacturer [optical density (OD) ≥0.75], the assay demonstrated limited diagnostic capacity with a sensitivity of 95% and a specificity of 41% compared with the Leptospira microscopic agglutination test, which is the serological gold standard. However, re-evaluation of the diagnostic cut-off to an OD of 1.7 demonstrated improved diagnostic accuracy overall (sensitivity 70%; specificity 78%).     

Predictive diagnostic value of the tourniquet test for the diagnosis of dengue infection in adults

Objective To examine the accuracy of the admission tourniquet test in the diagnosis of dengue infection among Lao adults. Methods Prospective assessment of the predictive diagnostic value of the tourniquet test for the diagnosis of dengue infection, as defined by IgM, IgG and NS1 ELISAs (Panbio Ltd, Australia), among Lao adult inpatients with clinically suspected dengue infection. Results Of 234 patients with clinically suspected dengue infection on admission, 73% were serologically confirmed to have dengue, while 64 patients with negative dengue serology were diagnosed as having scrub typhus (39%), murine typhus (11%), undetermined typhus (12%), Japanese encephalitis virus (5%), undetermined flavivirus (5%) and typhoid fever (3%); 25% had no identifiable aetiology. The tourniquet test was positive in 29.1% (95% CI = 23.2–34.9%) of all patients and in 34.1% (95% CI = 27.0–41.2%) of dengue‐seropositive patients, in 32.7% (95% CI = 23.5–41.8) of those with dengue fever and in 36.4% (95% CI = 24.7–48.0) of those with dengue haemorrhagic fever. Interobserver agreement for the tourniquet test was 90.2% (95% CI = 86.4–94.0) (Kappa = 0.76). Using ELISAs as the diagnostic gold standard, the sensitivity of the tourniquet test was 33.5–34%; its specificity was 84–91%. The positive and negative predictive values were 85–90% and 32.5–34%, respectively. Conclusions The admission tourniquet test has low sensitivity and adds relatively little value to the diagnosis of dengue among Lao adult inpatients with suspected dengue. Although a positive tourniquet test suggests dengue and that treatment of alternative diagnoses may not be needed, a negative test result does not exclude dengue.     

Evaluation of a Simple Blood Culture Amplification and Antigen Detection Method for Diagnosis of Salmonella enterica Serovar Typhi Bacteremia

In most areas where typhoid is endemic, laboratory diagnosis is not possible due to the lack of appropriate facilities. We investigated whether the combination of blood culture amplification of Salmonella enterica serovar Typhi with an S. Typhi antigen rapid diagnostic test (RDT) could be an accurate and inexpensive tool for the accelerated diagnosis of patients with acute typhoid in Laos. For a panel of 23 Gram-negative reference pathogens, the Standard Diagnostics (catalog no. 15FK20; Kyonggi-do, South Korea) RDT gave positive results for S. Typhi NCTC 8385, S. Typhi NCTC 786 (Vi negative), Salmonella enterica serovar Enteritidis (ATCC 13076), and Salmonella enterica serovar Ndolo NCTC 8700 (all group D). In a prospective study of 6,456 blood culture bottles from 3,028 patients over 15 months, 392 blood culture bottles (6.1%) from 221 (7.3%) patients had Gram-negative rods (GNRs) seen in the blood culture fluid. The sensitivity, negative predictive value, specificity, and positive predictive value were 96.7%, 99.5%, 97.9%, and 87.9%, respectively, for patients with proven S. Typhi bacteremia and 91.2%, 98.4%, 98.9%, and 93.9% for patients with group D Salmonella. The median (range) number of days between diagnosis by RDT and reference assays was 1 (−1 to +2) day for those with confirmed S. Typhi. The use of antigen-based pathogen detection in blood culture fluid may be a useful, relatively rapid, inexpensive, and accurate technique for the identification of important causes of bacteremia in the tropics.     

Detection of non-typhoid Salmonella infection by citrus and citrus extracts in Lao PDR

ObjectiveTo know the current state of non-typhoid Salmonella infection in Laos. To examine the usefulness of new screening methods for Salmonella using citrus.MethodsNon-typhoid Salmonella infection of person in Lao PDR was studied in this research (2004-2009). The site was Vientiane capital city in 2004. Research from rural villages locating suburb of Vientiane during 2005-2008 was carried out. Rural villages in Attapu province where ethnic minorities were living was searched for this study in 2009. During this research, to detect Salmonella strain, a new method using citrus and citrus extract named MY phenomenon that observing black ring (MIDO ring) on DHL agar was tried. The slice lemon and lime were used for this trial in 2004. After 2005, disk of ascorbic acid and citric acid were used for the device instead of citrus fruits itself.ResultsDuring this research, 65 of 272 human samples (23.9%) were infected with non-typhoid Salmonella.ConclusionsDuring this study, the method using citrus and citrus extracts was accepted for the detection of Salmonella. This study shows that with citrus and citrus extract, detection of Salmonella is possible using only DHL media. Results suggest that infectious rate of non-typhoid Salmonella was high.