Vincent P.K. Titanji

Low Anticoagulant Heparin Disrupts Plasmodium falciparum Rosettes in Fresh Clinical Isolates

The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria.

Malaria vectors and transmission dynamics in coastal south-western Cameroon

BackgroundMalaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas.MethodsA 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months.ResultsIn all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species.ConclusionMalaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa.

Acute Spotted Fever Rickettsiosis among Febrile Patients, Cameroon

Although potential arthropod vectors are abundant in Cameroon, acute febrile illnesses are rarely evaluated for arboviral or rickettsial infections. Serum samples from 234 acutely febrile patients at clinics in Tiko and Buea, Cameroon, were examined for antibodies to Rickettsia africae and African alphaviruses and flaviviruses. These serum samples did not contain antibodies against typhoid, and blood malarial parasites were not detected. Serum samples of 32% contained immunoglobulin M antibodies reactive with R. africae by immunofluorescence assay and were reactive with outer membrane proteins A and B of R. africae by immunoblotting. These findings established a diagnosis of acute rickettsiosis, most likely African tick-bite fever. Hemagglutination inhibition testing of the serum samples also detected antibodies to Chikungunya virus (47%) and flaviviruses (47%). High prevalence of antibodies to arboviruses may represent a major, previously unrecognized public health problem in an area where endemic malaria and typhoid fever have been the principal diagnostic considerations.

In Vitro Antiplasmodial Activity and Cytotoxicity of Extracts of Selected Medicinal Plants Used byTraditional Healers of Western Cameroon

Medicinal plants play a key role in malaria control in Africa, especially in remote areas where health facilities are limited. In order to assess their acclaimed potentials, eleven extracts were prepared from seven selected plants commonly used in Western Cameroon, and tested both for their antiplasmodial activity and cytotoxicity. The antiplasmodial activity was assessed using Lactate Dehydrogenase Assay (pLDH) and the cytotoxicity estimated on LLC-MK2 monkey kidney epithelial cells. Seven extracts from five different plants were significantly active, with very weak or no cytotoxicity. The Dacryodes edulis leaves showed the highest activity (IC50 of 6.45 μg/mL on 3D7 and 8.2 μg/mL on DD2) followed by the leaves of Vernonia amygdalina (IC50 of 8.72 and 11.27 μg/mL on 3D7 and DD2 resp.) and roots of V. amygdalina (IC50 of 8.72 μg/mL on 3D7), Coula edulis leaves (IC50 of 13.80 μg/mL and 5.79 μg/mL on 3D7 and DD2 resp.), Eucalyptus globulus leaves (IC50 of 16.80 μg/mL and 26.45 μg/mL on 3D7 and DD2) and Cuviera longiflora stem bark (IC50 of 20.24 μg/mL and 13.91 μg/mL on 3D7 and DD2). These findings justify the use of five of the seven plants in malaria treatment by traditional healers of Western Cameroon.

Molecular typing reveals substantial Plasmodium vivax infection in asymptomatic adults in a rural area of Cameroon

BackgroundMalaria in Cameroon is due to infections by Plasmodium falciparum and, to a lesser extent, Plasmodium malariae and Plasmodium ovale, but rarely Plasmodium vivax. A recent report suggested “Plasmodium vivax–like” infections around the study area that remained unconfirmed. Therefore, molecular and antigenic typing was used to investigate the prevalence of P. vivax and Duffy in asymptomatic adults resident in Bolifamba.MethodsA cross-sectional study was conducted from July 2008 to October 2009. The status of all parasite species was determined by nested PCR in 269 blood samples collected. The P. falciparum and P. vivax anti-MSP/CSP antibody status of each subject was also determined qualitatively by a rapid card assay. Parasite DNA was extracted from a sample infected with three parasite species, purified and sequenced. The Duffy antigen status of 12 subjects infected with P. vivax was also determined by sequencing. In silico web-based tools were used to analyse sequence data for similarities and matches to reference sequences in public DNA databases.ResultsThe overall malaria parasite prevalence in 269 individuals was 32.3% (87) as determined by PCR. Remarkably, 14.9% (13/87) of infections were caused either exclusively or concomitantly by P. vivax, established both by PCR and microscopic examination of blood smears, in individuals both positive (50%, 6/12) and negative (50%, 6/12) for the Duffy receptor. A triple infection by P. falciparum, P. vivax and P. malariae, was detected in one infected individual. Anti-MSP/CSP antibodies were detected in 72.1% (194/269) of samples, indicating high and continuous exposure to infection through mosquito bites.DiscussionThese data provide the first molecular evidence of P. vivax in Duffy positive and negative Cameroonians and suggest that there may be a significant prevalence of P. vivax infection than expected in the study area. Whether the P. vivax cases were imported or due to expansion of a founder effect was not investigated. Notwithstanding, the presence of P. vivax may complicate control efforts if these parasites become hypnozoitic or latent as the liver stage.ConclusionsThese data strongly suggest that P. vivax is endemic to the south-west region of Cameroon and should be taken into account when designing malaria control strategies.

Mycobacterium tuberculosis complex drug resistance pattern and identification of species causing tuberculosis in the West and Centre regions of Cameroon

BackgroundData on the levels of resistance of Mycobacterium tuberculosis complex (MTBC) strains to first line anti-tuberculosis drugs in Cameroon, and on the species of MTBC circulating in the country are obsolete. The picture about 10 years after the last studies, and 6 years after the re-organisation of the National Tuberculosis (TB) Control Programme (NTBCP) is not known.MethodsThe study was conducted from February to July 2009 in the West and Centre regions of Cameroon. A total of 756 suspected patients were studied. MTBC species were detected by the standard Ziehl-Neelsen staining method. Bacterial susceptibility to the first line drugs [isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (SM)] were performed on cultures using the indirect proportion method. MTBC species were identified by standard biochemical and culture methods.ResultsOf the 756 suspected patients, 154 (20.37%) were positive by smear microscopy. Of these, 20.77% were HIV patients. The growth of Mycobacterium was observed with the sputa from 149 (96.75%) subjects. All the isolates were identified as either M. tuberculosis or M. africanum. Among these, 16 (10.73%) were resistant to at least one drug (13.3% for the West region and 8.1% for the Centre). The initial resistance rates were 7.35% for the Centre region and 11.29% for the West region, while the acquired resistance rates were 16.66% (1/6) for the Centre region and 23.07% (3/13) for the West. Within the two regions, the highest total resistance to one drug was obtained with INH and SM (2.68% each). Multidrug-resistance (MDR) was observed only in the West region at a rate of 6.67%. No resistance was recorded for EMB.ConclusionsM. tuberculosis and M. africanum remain the MTBC species causing pulmonary TB in the West and Centre regions of Cameroon. Following the re-organisation of the NTBCP, resistance to all first line anti-TB drugs has declined significantly (p < 0.05 for West; and p < 0.01 for Centre) in comparison to previous studies. However, the general rates of anti-TB drug resistance remain high in the country, underscoring the need for greater enforcement of control strategies.

HLA-DQ haplotypes in 15 different populations

In order to understand the forces governing the evolution of the DQ molecule, PCR-based methods have been used to type the DQA1 and DQB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including Africans, Asians, Amerindians and Caucasians. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger populations where admixture has occurred. Nine DQA1 alleles and 18 DQB1 alleles have been identified which make up 42 distinct DQ haplotypes. Some haplotypes are found in all ethnic groups while others are confined to a single ethnic group or population. Despite evidence of recombination between the DQA1 and DQB1 loci, there are no examples of a haplotype carrying a DQw1-associated alpha chain and a DQw2-, DQw3-, or DQw4-associated beta chain in cis (and vice versa). These data suggest that these haplotypes, which encode unstable heterodimers, are rapidly removed from the population through natural selection.

Mycobacterium tuberculosis is the causative agent of tuberculosis in the southern ecological zones of Cameroon, as shown by genetic analysis

BackgroundTuberculosis (TB) is a major cause of mortality and suffering worldwide, with over 95% of TB deaths occurring in low- and middle-income countries. In recent years, molecular typing methods have been widely used in epidemiological studies to aid the control of TB, but this usage has not been the case with many African countries, including Cameroon. The aims of the present investigation were to identify and evaluate the diversity of the Mycobacterium tuberculosis complex (MTBC) isolates circulating in two ecological zones of Cameroon, seven years after the last studies in the West Region, and after the re-organization of the National TB Control Program (NTBCP). These were expected to shed light also on the transmission of TB in the country. The study was conducted from February to July 2009. During this period, 169 patients with symptomatic disease and with sputum cultures that were positive for MTBC were randomly selected for the study from amongst 964 suspected patients in the savannah mosaic zone (West and North West regions) and the tropical rainforest zone (Central region). After culture and diagnosis, DNA was extracted from each of the MTBC isolates and transported to the BecA-ILRI Hub in Nairobi, Kenya for molecular analysis.MethodsGenetic characterization was done by mycobacterial interspersed repetitive unit–variable number tandem repeat typing (MIRU-VNTR) and Spoligotyping.ResultsMolecular analysis showed that all TB cases reported in this study were caused by infections with Mycobacterium tuberculosis (98.8%) and Mycobacterium africanum (M. africanum) (1.2%) respectively. We did not detect any M. bovis. Comparative analyses using spoligotyping revealed that the majority of isolates belong to major clades of M. tuberculosis: Haarlem (7.6%), Latin American-Mediterranean (34.4%) and T clade (26.7%); the remaining isolates (31.3%) where distributed among the minor clades. The predominant group of isolates (34.4%) corresponded to spoligotype 61, previously described as the “Cameroon family. Further analysis based on MIRU-VNTR profiles had greater resolving power than spoligotyping and defined additional genotypes in the same spoligotype cluster.ConclusionThe molecular characterization of MTBC strains from humans in two ecological regions of Cameroon has shown that M. tuberculosis sensu stricto is the predominant agent of TB cases in the zones. Three decades ago, TB was reported to be caused by M. africanum in 56.0% of cases. The present findings are consistent with a major shift in the prevalence of M. tuberculosis in Cameroon.

Ethnobotanical survey and in vitro antiplasmodial activity of medicinal plants used to treat malaria in Kagera and Lindi regions, Tanzania

Tanzania has over 12,000 plant species, some of which are endemic and have potential to yield useful medicines. This study seeks to document such plants used as traditional medicines for treatment of malaria in Kagera region of northwestern Tanzania and Lindi region in south eastern Tanzania. The study also reports on the antiplasmodial activity against chloroquine-resistant Plasmodium falciparum (Dd2) strain of some of the documented plants using the parasite lactate dehydrogenase method. A total of 108 plant species, among which the families Compositae (14; 12.96%), Fabaceae (12; 11.11%), Euphorbiaceae (8; 7.41%), Melastomataceae (6; 5.56%) and Myrtaceae (4; 3.70%) were documented. Sixteen (16; 44.4%) of 36 extracts from 31 plant species that were tested inhibited malaria parasites growth by more than 50%. Bersema abyssinica stem bark extract was the most active with 86.67% inhibition rate followed by Bridelia micrantha stem bark extract with 71.87% inhibition rate. These results confirm the potential for plants used in traditional medicine to yield active antimalarial compounds. Further in vitro and in vivo screening supported by bioassay-guided isolation of active compounds from plants showing good safety margin is suggested.

Evaluation of the cytotoxic activity of extracts from medicinal plants used for the treatment of malaria in Kagera and Lindi regions, Tanzania

A number of medicinal plants used for treatment of malaria in Tanzania have been documented, but information on their safety and efficacy is still based on traditional knowledge accumulated over years and not on pre-clinical and clinical evaluation. The present study aimed to assess the cytotoxic activity of extracts of selected plant species used for treatment of malaria in Tanzania. Ethanol extracts were evaluated for cytoxicity by using MTT assay on LLC-MK2 cells and by brine shrimp lethality assay. Forty five (93.75%) out of 48 crude extracts assessed using LLC-MK2 cells were non-cytotoxic while three extracts (6.25%) were cytotoxic with CC50