Polymerase Chain Reaction Used To Describe Larval Habitat Use by Anopheles gambiae Complex (Diptera: Culicidae) in the Environs of Ifakara, Tanzania

Larvae of the Anopheles gambiae complex were collected in and around the town of Ifakara, southern Tanzania during the wet season of 1994 and identified to species by polymerase chain reaction. All but 1 surface pool contained mixed populations of An. gambiae and An. arabiensis larvae. The 2 species varied among locations rather than types of water. An. arabiensis predominated in pools close to cattle. The numbers of identified early instars of both species were similar, but more An. gambiae 4th instars were identified, perhaps indicating that An. gambiae were able to survive heavy rainfall better than A. arabiensi

Comparison of three methods for detection of Plasmodium oocysts in wild-caught mosquitoes

One hundred forty-nine Anopheles gambicie s.l. and 260 A. funestus were collected from the field in Namawala, Tanzania in June 1994. The mosquitoes were dissected and examined for plasmodium oocysts using two microscopic techniques and the polymerase chain reaction (PCR) method. The oldest and simplest technique, the saline test, detected oocysts in 94 out of the 97 mosquitoes found to have oocysts by the more sensitive merbromin stain and PCR techniques. The saline test was found to be a sensitive and reliable field technique for detection of large Plasmodium oocysts in mosquitoes. The merbromin staining technique was as sensitive as PCR, detecting even small oocyst

Investigation of the Risk of Infection of Urinary Schistosomiasis at Mahem and Galilea Communities in the Greater Accra Region of Ghana

Urinary schistosomiasis is of great public health importance in developing countries. It has adverse economic and health implications on residents living in endemic areas. Various factors including human behaviour are known to play key role in the transmission of the disease. The knowledge of the levels of risk of infection of urinary schistosomiasis and people’s perception will be an important tool in its control. The study determined the prevalence of urinary schistosomiasis and the risk of infection in some communities near the Weija lake in the Ga District. It assessed the knowledge base of the subjects on the disease and its impact on transmission. Data were collected on demographic variables, some behavioural activities in water bodies, knowledge base on the disease and sanitary facilities. Urine samples were analysed using the centrifugation technique. The percentage prevalence for Mahem and Galilea were 58% and 49%, respectively. The difference in prevalence was insignificant; 0.09 (-0.04, 0.21; P < 0.426). Bloody urine was associated with high risk of infection; OR of 4.55 (2.82, 7.36); P < 0.001. Subjects with primary level of education and invariably below 26 years of age had about two times the risk of infection; OR of 2.12 (1.13, 3.97); P < 0.02. The communities had 52% prevalence of urinary schistosomiasis. Frequent contacts and use of the infested lake were associated with infection. Educational intervention alone may not be effective in the control of the disease. The use of an integrated approach should be given favourable consideration

Herb-drug interaction: Effect of aqueous extract of Bridelia ferruginea leaves on the pharmacokinetics of metformin

The concurrent use of herbal medicines and orthodox drugs, especially in the treatment and management of chronic ailments, may result in clinically significant herb-drug interactions. Bridelia ferruginea Benth (Euphorbiaceae) is a common medicinal plant with known anti-diabetic properties and has been reported to be taken alongside the orthodox medicine, metformin. The aim of this study was to investigate the effect of aqueous extract of B. ferruginea leaves on the pharmacokinetics of metformin in female Sprague-Dawley rats. Reconstituted freeze dried extract of B. ferruginea leaves (30 mg/kg), and metformin (7 mg/kg) were administered concurrently as a single dose to female Sprague-Dawley rats. Whole blood samples (1 ml) were aseptically withdrawn by tail bleeding at 1, 2, 4, 8, and 24 h after administration of the single dose for pharmacokinetics analyses. Concurrent administration of metformin and B. ferruginea significantly affected (P &lt; 0.05) all the pharmacokinetics parameters of metformin except for the time to attain the maximum concentration (Tmax), which increased but insignificantly. Whereas the area under the curve, maximum whole blood concentration (Cmax) and half-life (T½) of metformin decreased significantly in the presence of B. ferruginea, the elimination rate constant (Kel), clearance (Cl), absorption rate constant (Ka), and volume of distribution (Vd) of metformin increased significantly in the presence of B. ferruginea. Therefore, in clinical practice, patients should be advised on the implication of concurrent administration of metformin and B. ferrruginea

Sero-epidemiology of toxoplasmosis amongst pregnant women in the greater Accra region of Ghana

Objectives: To investigate Toxoplasma infection among pregnant women in relation to exposure to infection risk, age and pregnancy-related risk factors. Design and Methods: This cross-sectional study involved 294 pregnant women attending ante-natal clinic in Accra who consented to participate. Personal and Toxoplasma infection risk related data were obtained by questionnaire interviews. Venous blood was safely drawn from each participant and spun to obtain sera.Each of the 159 randomly selected serum samples was tested for specific anti-Toxoplasma (anti-T. gondii) antibodies IgG, IgA and IgM using a commercial ELISA kit (Calbiotech Inc., CA). ELISA results were correlated with exposure to possible infection risk factorsas well as age and pregnancy-related risk factors. Results: The 159 women aged 15-40 years in their first, second and third trimesters, numbered 29, 70 and 60, respectively. An overall anti-T. gondii antibodies IgG, IgA and IgM seroprevalence of 92.5% (147/159)was recorded, with 4.1% (6/147) of them having anti- IgG only. The remaining 88.7% (141/159) had anti- Toxoplasma antibodies IgG, IgA and IgM in various combinations and consisted of 17.7% (25/141) in their first, 44.0% (62/141) in their second, and 38.3% (54/141) in their third, trimesters. Twelve women (7.6%) were seronegative for all 3 antibodies Conclusions: Seroprevalence was high among the women and exposure to contact with cats’ faeces was found to be the major T. gondii infection risk factor. Age and pregnancy-related risk factors did not have association with T. gondii infection within the limitationsof this study

6. Multiple Plasmodium falciparum infections in Tanzanian infants

Paired blood samples from 99 Tanzanian infants were analysed to examine the infection dynamics of Plasmodium falciparum during the first year of life. Infecting parasites were genotyped by polymerase chain reaction amplification of the polymorphic gene for the merozoite surface protein 2 and subsequent analysis according to the resulting restriction fragment length polymorphism pattern. The same samples served as controls in a parallel case-control study for which an additional blood sample was taken from each child during a fever episode. The relationship of the number of concurrent infections (multiplicity) with age and morbidity was analysed and results were compared to those of a similar study on older children between 2 and 7 years of age, carried out in the same village at the same time. The mean of 2 infecting genotypes per positive blood sample from community surveys was low compared to that in older children, and there was no significant age-dependency of multiplicity within the first year of life. Multiplicity of infection in fever cases was also independent of age. In infants, multiplicity was positively associated with parasite density and risk of clinical malaria, in contrast to the situation in older children (>2 years). The findings help in the understanding of infection dynamics, premunition, and development of semi-immunity in malari

9. Effect of insecticide-treated bed nets on haemoglobin values, prevalence and multiplicity of infection with Plasmodium falciparum in a randomized controlled trial in Tanzania

A randomized controlled trial of insecticide-treated bed nets (ITNs) was conducted in an area of high malaria transmission in Tanzania in order to assess the effects of ITNs on infection and anaemia. One hundred and twenty-two children, aged 5 to 24 months, were randomly allocated to 2 groups, one of which received ITNs. Outcome measures were assessed in 6 consecutive months with monthly cross-sectional surveys. These measures were haemoglobin values, Plasmodium falciparum prevalence and density, and multiplicity of infection determined by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) of the msp2 locus. There was a significant increase in mean heamoglobin values and a significant decrease of 16·4% in microscopically determined P. falciparum prevalence in children in the ITN group six months after the start of the trial. Both effects were more pronounced in younger children. However, no significant difference was observed in parasite density or multiplicity of infection among infected children. Comparison with PCR results indicated that microscopically subpatent parasitaemia was more frequently found in children in the ITN group. This, together with the observed similar multiplicity in the 2 groups, suggests that infections are maintained despite ITN use, owing to the chronicity of infections. This study shows that ITNs reduce the risk of anaemia in highly exposed young children. The virtually unchanged multiplicity of infection indicates that the potentially protective concomitant immunity is not compromise

Community coverage of an antimalarial combination of artesunate and amodiaquine in Makamba Province, Burundi, nine months after its introduction

BACKGROUND: In 2003, artesunate-amodiaquine (AS+AQ) was introduced as the new first-line treatment for uncomplicated malaria in Burundi. After confirmed diagnosis, treatment was delivered at subsidized prices in public health centres. Nine months after its implementation a study was carried out to assess whether children below five years of age with uncomplicated malaria were actually receiving AS+AQ. METHODS: A community-based study was conducted in Makamba province. Randomly selected households containing one or more children under five with reported fever onset within fourteen days before the study date were eligible. Case-management information was collected based on caregiver recall. A case definition of symptomatic malaria from observations of children presenting a confirmed malaria episode on the day of the survey was developed. Based on this definition, those children who had probable malaria among those with fever onset in the 14 days prior to the study were identified retrospectively. Treatment coverage with AS+AQ was then estimated among these probable malaria cases. RESULTS: Out of 195 children with fever on the day of the study, 92 were confirmed as true malaria cases and 103 tested negative. The combination of 'loss of appetite', 'sweating', 'shivering' and 'intermittent fever' yielded the highest possible positive predictive value, and was chosen as the case definition of malaria. Out of 526 children who had had fever 14 days prior to the survey, 165 (31.4%) were defined as probable malaria cases using this definition. Among them, 20 (14.1%) had been treated with AS+AQ, 10 with quinine (5%), 68 (41%) received non-malaria treatments, and 67 got traditional treatment or nothing (39.9%). Most people sought treatment from public health centres (23/99) followed by private clinics (15/99, 14.1%). The median price paid for AS+AQ was 0.5 US$. CONCLUSION: AS+AQ was the most common treatment for patients with probable malaria at public health centres, but coverage was low due to low health centre utilisation and apparently inappropriate prescribing. In addition, AS+AQ was given to patients at a price ten times higher than the subsidized price. The availability and proper use of ACTs should be monitored and maximized after their introduction in order to have a significant impact on the burden of malaria

Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR

<p>Abstract</p> <p>Background</p> <p>This study describes the use of thick blood films (TBF) as specimens for DNA amplification with the <it>Plasmodium </it>species-specific real-time PCR that was recently validated on whole blood samples.</p> <p>Methods</p> <p>The panel of 135 Giemsa-stained clinical TBFs represented single infections of the four <it>Plasmodium </it>species with varying parasite densities or only gametocytes, mixed infections, and negative samples and was stored for up to 12 years. Half of the Giemsa-stained TBF was scraped off by a sterile scalpel and collected into phosphate buffered saline. DNA was extracted with the Qiagen DNA mini kit with minor modifications. DNA was amplified with the 18S rRNA real-time PCR targeting the four <it>Plasmodium </it>species with four species-specific primers and probes in combination with one genus-specific reverse primer. Results of the PCR on TBF were compared to those of the PCR on whole blood and to microscopy.</p> <p>Results</p> <p>Correct identification for single species infections was obtained for all TBF samples with <it>Plasmodium falciparum </it>(n = 50), <it>Plasmodium vivax </it>(n = 25), <it>Plasmodium ovale </it>(n = 25) and in all but one samples with <it>Plasmodium malariae </it>(n = 10). Compared to whole blood samples, higher Ct-values were observed by PCR on TBF with a mean difference of 5.93. Four out of five mixed infections were correctly identified with PCR on TBF. None of the negative samples (n = 20) gave a PCR signal. PCR on TBF showed a detection limit of 0.2 asexual parasites/μl compared to 0.02/μl for whole blood. Intra-run variation was higher for PCR on TBF (%CV 1.90) compared to PCR on whole blood (%CV 0.54). Compared to microscopy, PCR on TBF generated three more species identifications in samples containing a single species and detected the same four mixed-infections.</p> <p>Conclusions</p> <p>Giemsa-stained TBFs are a reliable source of DNA for <it>Plasmodium </it>real-time PCR analysis, allowing applications in reference and research settings in case whole blood samples are not available.</p

Using rapid diagnostic tests as source of malaria parasite DNA for molecular analyses in the era of declining malaria prevalence

BACKGROUND: Malaria prevalence has recently declined markedly in many parts of Tanzania and other sub-Saharan African countries due to scaling-up of control interventions including more efficient treatment regimens (e.g. artemisinin-based combination therapy) and insecticide-treated bed nets. Although continued molecular surveillance of malaria parasites is important to early identify emerging anti-malarial drug resistance, it is becoming increasingly difficult to obtain parasite samples from ongoing studies, such as routine drug efficacy trials. To explore other sources of parasite DNA, this study was conducted to examine if sufficient DNA could be successfully extracted from malaria rapid diagnostic tests (RDTs), used and collected as part of routine case management services in health facilities, and thus forming the basis for molecular analyses, surveillance and quality control (QC) testing of RDTs. METHODS: One hyper-parasitaemic blood sample (131,260 asexual parasites/μl) was serially diluted in triplicates with whole blood and blotted on RDTs. DNA was extracted from the RDT dilution series, either immediately or after storage for one month at room temperature. The extracted DNA was amplified using a nested PCR method for Plasmodium species detection. Additionally, 165 archived RDTs obtained from ongoing malaria studies were analysed to determine the amplification success and test applicability of RDT for QC testing. RESULTS: DNA was successfully extracted and amplified from the three sets of RDT dilution series and the minimum detection limit of PCR was <1 asexual parasite/μl. DNA was also successfully amplified from (1) 70/71 (98.6%) archived positive RDTs (RDTs and microscopy positive) (2) 52/63 (82.5%) false negative RDTs (negative by RDTs but positive by microscopy) and (3) 4/24 (16.7%) false positive RDTs (positive by RDTs but negative by microscopy). Finally, 7(100%) negative RDTs (negative by RDTs and microscopy) were also negative by PCR. CONCLUSION: This study showed that DNA extracted from archived RDTs can be successfully amplified by PCR and used for detection of malaria parasites. Since Tanzania is planning to introduce RDTs in all health facilities (and possibly also at community level), availability of archived RDTs will provide an alternative source of DNA for genetic studies such as continued surveillance of parasite resistance to anti-malarial drugs. The DNA obtained from RDTs can also be used for QC testing by detecting malaria parasites using PCR in places without facilities for microscopy