Ib C. Bygbjerg

Accuracy of malaria rapid diagnostic tests in community studies and their impact on treatment of malaria in an area with declining malaria burden in north-eastern Tanzania

BackgroundDespite some problems related to accuracy and applicability of malaria rapid diagnostic tests (RDTs), they are currently the best option in areas with limited laboratory services for improving case management through parasitological diagnosis and reducing over-treatment. This study was conducted in areas with declining malaria burden to assess; 1) the accuracy of RDTs when used at different community settings, 2) the impact of using RDTs on anti-malarial dispensing by community-owned resource persons (CORPs) and 3) adherence of CORPs to treatment guidelines by providing treatment based on RDT results.MethodsData were obtained from: 1) a longitudinal study of passive case detection of fevers using CORPs in six villages in Korogwe; and 2) cross-sectional surveys (CSS) in six villages of Korogwe and Muheza districts, north-eastern, Tanzania. Performance of RDTs was compared with microscopy as a gold standard, and factors affecting their accuracy were explored using a multivariate logistic regression model.ResultsOverall sensitivity and specificity of RDTs in the longitudinal study (of 23,793 febrile cases; 18,154 with microscopy and RDTs results) were 88.6% and 88.2%, respectively. In the CSS, the sensitivity was significantly lower (63.4%; χ2 = 367.7, p < 0.001), while the specificity was significantly higher (94.3%; χ2 = 143.1, p < 0.001) when compared to the longitudinal study. As determinants of sensitivity of RDTs in both studies, parasite density of < 200 asexual parasites/μl was significantly associated with high risk of false negative RDTs (OR≥16.60, p < 0.001), while the risk of false negative test was significantly lower among cases with fever (axillary temperature ≥37.5°C) (OR ≤ 0.63, p ≤ 0.027). The risk of false positive RDT (as a determinant of specificity) was significantly higher in cases with fever compared to afebrile cases (OR≥2.40, p < 0.001). Using RDTs reduced anti-malarials dispensing from 98.9% to 32.1% in cases aged ≥5 years.ConclusionAlthough RDTs had low sensitivity and specificity, which varied widely depending on fever and parasite density, using RDTs reduced over-treatment with anti-malarials significantly. Thus, with declining malaria prevalence, RDTs will potentially identify majority of febrile cases with parasites and lead to improved management of malaria and non-malaria fevers.

IgG reactivities against recombinant Rhoptry-Associated Protein-1 (rRAP-1) are associated with mixed Plasmodium infections and protection against disease in Tanzanian children

A cross-sectional sero-epidemiological study was performed in Magoda, Tanzania, an area where malaria is holoendemic. Blood samples were collected from children (1-4 years) and tested for IgG antibody reactivity against 2 recombinant protein fragments of Plasmodium falciparum Rhoptry-Associated Protein-1 (rRAP-1). The data were related to the prevalence of malarial disease and single P. falciparum or mixed Plasmodium infections. Fever (> or = 37.5 degrees C) in combination with parasite densities > 5000/microliter were used to distinguish between children with asymptomatic malaria infections and those with acute clinical disease. Furthermore, C-reactive protein (CRP) was applied as a surrogate marker of malaria morbidity. The prevalence of Plasmodium infections was 96.0%. Eleven children were defined as clinical malaria cases, all with single P. falciparum infections. The density of P. falciparum was significantly lower in children with mixed Plasmodium infections compared to those with single P. falciparum infections. Children with asymptomatic P. falciparum infections had higher IgG reactivities to rRAP-1, compared to IgG reactivities of children with malarial disease. Children with mixed Plasmodium infections generally showed elevated IgG reactivity to rRAP-1, when compared to children with single P. falciparum infections. The possible relationship between mixed species infections, clinical outcome of the disease and antibody responses to RAP-1 is discussed.

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

BackgroundMalaria 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.MethodsOne 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.ResultsDNA 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.ConclusionThis 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.

Laboratory indicators of the diagnosis and course of imported malaria

When travellers return from malaria-endemic areas and present to hospital with fever, microscopy of blood smears remains the leading method to verify a suspected diagnosis of malaria. Additional laboratory abnormalities may, however, also be indicative of acute malaria infection. We monitored prospectively a group of patients with imported Plasmodium falciparum (n =28) or P. vivax/P. ovale (n =12) infection, respectively, and assessed haemoglobin, leucocytes, thrombocytes, C-reactive protein, coagulation factor II-VII-X, lactate dehydrogenase and bilirubin during 7 d of admission and weekly until d 28. For comparison, admission values of a group of febrile patients with suspected malaria, but with negative blood slides, were also assessed (n =66). The thrombocyte, leucocyte counts and coagulation factor II-VII-X were significantly lower in the malaria group compared to the non-malaria group, whereas the C-reactive protein, lactate dehydrogenase and bilirubin were significantly higher in the malaria group. The differences were particularly strong with falciparum malaria. By contrast, haemoglobin levels were not affected. In conclusion, our study emphasizes the role of a few commonly analysed laboratory parameters, in particular thrombocyte counts, in guiding the clinician managing a returning traveller with fever.

Development and evaluation of a multiplex screening assay for Plasmodium falciparum exposure

Transfusion transmitted malaria (TTM) in non-endemic countries is reduced by questioning blood donors and screening of donated blood. Conventional screening is performed by Indirect Fluorescence Antibody Test (IFAT). This method is manual and difficult to standardize. Here we study the diagnostic performance of a multiplex assay for detection of antibodies against Plasmodium falciparum in donor blood using IFAT as a comparator. A multiplex assay (MPA) containing the antigens GLURP-R0, GLURP-R2, MSP3, MSP1 hybrid and AMA1 was constructed using xMAP® technology. A discrimination index for exposure to P. falciparum malaria was calculated by comparing travelers with clinical malaria (n=52) and non-exposed blood donors (n=119). The index was evaluated on blood donors with suspected malaria exposure (n=249) and compared to the diagnostic performance of IFAT. At a specificity of 95.8 %, the MPA discrimination index exhibited a diagnostic sensitivity of 90.4 % in travelers hospitalized with malaria. Percent agreement with IFAT was 92.3 %. Screening plasma from blood donors with suspected malaria exposure, we found 4.8 % to be positive by IFAT and 5.2 % by MPA with an agreement of 93.2 %. The calculated index from the MPA exhibits similar diagnostic performance as IFAT for detection of P. falciparum malaria. Combining the antibody response against multiple antigens in a discrimination index increased the sensitivity of the MPA and reduced the readout to a single value.

Development of ELISA-based methods to measure the anti-malarial drug chloroquine in plasma and in pharmaceutical formulations.

BackgroundIn Central and South America and Eastern and Southern Africa, Plasmodium vivax infections accounts for 71-81% and 5% of malaria cases, respectively. In these areas, chloroquine (CQ) remains the treatment of choice for P. vivax malaria. In addition, CQ has recently proven to be an effective HIV-1 therapeutic agent. There is a dire need to continue monitoring quality of CQ as there is a major influx of substandard and fake formulations into malaria-endemic countries. The use of fake/substandard drugs will result in sub-therapeutic levels endangering the patient and possibly select for parasite resistance. The aim of this study was to develop an inexpensive, simple antibody-based ELISA to measure CQ concentrations in tablets and in plasma.MethodsA monoclonal antibody (MAb) that reacts with the N-side chain of the CQ molecule was prepared by use of a CQ analogue. A specific and reliable ELISA for detection of CQ was developed. The developed assay was validated by measuring CQ in tablets sold in Denmark, India and Sudan. Furthermore, kinetics of CQ concentrations in plasma of four volunteers, who ingested two tablets of Malarex® containing, 250 mg CQ base, were measured before drug intake, three hours later and thereafter at days 1, 3, 7, 14, 21 and 28. The same plasma samples were simultaneously measured by high performance liquid chromatography (HPLC).ResultsThe ELISA proved an easy-to-handle and very sensitive tool for the detection of CQ with a lower limit of detection at 3.9 ng/ml. ELISA levels of CQ in plasma showed high agreement with the levels obtained by HPLC (r = 0.98). The specificity in the negative control group was 100%.ConclusionThe developed ELISA can be used for quality screening of CQ in pharmaceutical formulations and for drug monitoring in malaria and in other infectious diseases, such as HIV, where CQ proved to be an effective therapeutic agent. The methodology has been exploited to develop monoclonal antibodies for the drugs used in artemisinin-based combination therapy (ACT).

Effect of folic and folinic acid on cytopenia occurring during co-trimoxazole treatment of Pneumocystis carinii pneumonia

12 AIDS/ARC patients with or suspected of Pneumocystis carinii pneumonia were treated with co-trimoxazole and received supplementary folic or folinic acid to avoid peripheral blood cytopenia. Most patients developed decreased numbers of neutrophils and hemoglobin while receiving co-trimoxazole. Supplementary folate/folinate could not abolish the drug-induced cytopenia. Routine prescription of folinic acid is not recommended. Folic acid is cheap and may be beneficial and should be prescribed.

Yield of diagnostic tests for opportunistic infections in AIDS: a survey of 33 patients.

To examine the therapeutic consequences of diagnostic tests for AIDS-related infections, case records from 33 deceased AIDS patients were reviewed; 23 were autopsied. Determination of serum antibody titres was not important. In particular, there was no relation between titres and isolation of cytomegalovirus (CMV); isolation attempts or possibly antigen determination would be better. Samples for CMV isolation were obtained from 31 patients; only 7 were negative. Blood, faeces, and particularly sputum cultures gave a low yield; the number of such examinations could be reduced considerably. However, 4/7 disseminated infections with atypical mycobacteria were only revealed at autopsy, despite numerous cultures in vivo. Liver biopsies were not helpful. Diagnostic procedures for Pneumocystis carinii pneumonia by lung biopsy caused pneumothorax in 3/15 patients; bronchoalveolar lavage or treatment/prophylaxis without diagnosis could be considered. In 8 autopsies, microscopy was suggestive of cerebral toxoplasmosis, but only 1 patient had presented important clinical symptoms. We suggest a schedule with regular microbiologic and parasitic examinations and few antibody tests, but with more antigen tests.