Genotyping of <it>Plasmodium falciparum</it> using antigenic polymorphic markers and to study anti-malarial drug resistance markers in malaria endemic areas of Bangladesh

<p>Abstract</p> <p>Background</p> <p>In the past many regions of Bangladesh were hyperendemic for malaria. Malaria control in the 1960s to 1970s eliminated malaria from the plains but in the Chittagong Hill Tracts remained a difficult to control reservoir. The Chittagong Hill Tracts have areas with between 1 and 10% annual malaria rates, predominately 90-95% <it>Plasmodium falciparum</it>. In Southeast Asia, multiplicity of infection for hypo-endemic regions has been approximately 1.5. Few studies on the genetic diversity of <it>P. falciparum</it> have been performed in Bangladesh. Anderson <it>et al.</it> performed a study in Khagrachari, northern Chittagong Hill Tracts in 2002 on 203 patients and found that parasites had a multiplicity of infection of 1.3 by MSP-1, MSP-2 and GLURP genotyping. A total of 94% of the isolates had the K76T <it>Pfcrt</it> chloroquine resistant genotype, and 70% showed the N86Y <it>Pfmdr1</it> genotype. Antifolate drug resistant genotypes were high with 99% and 73% of parasites having two or more mutations at the <it>dhfr</it> or <it>dhps</it> loci.</p> <p>Methods</p> <p>Nested and real-time polymerase chain reaction (PCR) methods were used to genotype <it>P. falciparum</it> using antigenic polymorphic markers and to study anti-malarial drug resistance markers in malaria endemic areas of Bangladesh.</p> <p>Results</p> <p>The analysis of polymorphic and drug resistant genotype on 33 paired recrudescent infections after drug treatment in the period 2004 to 2008 in the Chittagong Hill Tracts, which is just prior to countrywide provision of artemisinin combination therapy. Overall the multiplicity of infection for MSP-1 was 2.7 with a slightly smaller parasite diversity post-treatment. The 13 monoclonal infections by both GLURP and MSP-1 were evenly divided between pre- and post-treatment. The MSP-1 MAD block was most frequent in 66 of the samples. The prevalence of the K76T PfCRT chloroquine resistant allele was approximately 82% of the samples, while the resistant Pfmdr1 N86Y was present in 33% of the samples. Interestingly, the post-treatment samples had a small but significantly higher frequency of the sensitive PfCRT alleles by RT-PCR.</p> <p>Conclusion</p> <p>The parasite population retains high population diversity despite hypo-endemic transmission with retention, but decrease in the chloroquine-resistant allele and Pfmdr1 resistant alleles in the Chittagong Hill Tracts of Bangladesh.</p

Prevalence and characteristics of escherichia coli isolates harbouring shiga toxin genes (STX) from acute diarrhoeal patients in Dhaka, Bangladesh

Shiga toxin genes (stx) harbouring Escherichia coli (STEC) strains were isolated and identified from diarrhoeal patients visiting the Dhaka Hospital of ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh. Of the 189 E. coli strains isolated from 775 diarrhoeal stool specimens, 19 harboured stx1, and one isolate was revealed to have amplicons for both stx1 and stx2 by a PCR assay. Sequence analysis of the 349-bp stx1 from representative isolates revealed 100% homology with the sequence of stx1 available in the GenBank. Among the stx1 positive isolates, two harboured the eae but none were positive for hlyA, katP, etpD or saa genes. Fifteen of the 20 stx positive strains could be categorized into 13 non-O157 serogroups while 4 were untypable and one was a rough strain. Most of the STEC strains were resistant to ampicillin, cephalothin, co-trimoxazole, tetracycline, and nalidixic acid. In the Vero cell assay, all the strains were negative for expression of Shiga toxin (Stx). Randomly amplified polymorphic DNA (RAPD) PCR analysis demonstrated genetic diversity. This is one of the first reports to show the presence of STEC in diarrhoeal patients in Bangladesh

Characterising the effect of antimalarial drugs on the maturation and clearance of murine blood-stage Plasmodium parasites in vivo

The artemisinins are the first-line therapy for severe and uncomplicated malaria, since they cause rapid declines in parasitemia after treatment. Despite this, in vivo mechanisms underlying this rapid decline remain poorly characterised. The overall decline in parasitemia is the net effect of drug inhibition of parasites and host clearance, which competes against any ongoing parasite proliferation. Separating these mechanisms in vivo was not possible through measurements of total parasitemia alone. Therefore, we employed an adoptive transfer approach in which C57BL/6J mice were transfused with Plasmodium berghei ANKA strain-infected, fluorescent red blood cells, and subsequently drug-treated. This approach allowed us to distinguish between the initial drug-treated generation of parasites (Gen), and their progeny (Gen). Artesunate efficiently impaired maturation of Gen parasites, such that a sufficiently high dose completely arrested maturation after 6 h of in vivo exposure. In addition, artesunate-affected parasites were cleared from circulation with a half-life of 6.7 h. In vivo cell depletion studies using clodronate liposomes revealed an important role for host phagocytes in the removal of artesunate-affected parasites, particularly ring and trophozoite stages. Finally, we found that a second antimalarial drug, mefloquine, was less effective than artesunate at suppressing parasite maturation and driving host-mediated parasite clearance. Thus, we propose that in vivo artesunate treatment causes rapid decline in parasitemia by arresting parasite maturation and encouraging phagocyte-mediated clearance of parasitised RBCs

Malaria hotspots drive hypoendemic transmission in the Chittagong Hill Districts of Bangladesh.

Malaria is endemic in 13 of 64 districts of Bangladesh, representing a population at risk of about 27 million people. The highest rates of malaria in Bangladesh occur in the Chittagong Hill Districts, and Plasmodium falciparum (predominately chloroquine resistant) is the most prevalent species.The objective of this research was to describe the epidemiology of symptomatic P. falciparum malaria in an area of Bangladesh following the introduction of a national malaria control program. We carried out surveillance for symptomatic malaria due to P. falciparum in two demographically defined unions of the Chittagong Hill Districts in Bangladesh, bordering western Myanmar, between October 2009 and May 2012. The association between sociodemographics and temporal and climate factors with symptomatic P. falciparum infection over two years of surveillance data was assessed. Risk factors for infection were determined using a multivariate regression model.472 cases of symptomatic P. falciparum malaria cases were identified among 23,372 residents during the study period. Greater than 85% of cases occurred during the rainy season from May to October, and cases were highly clustered geographically within these two unions with more than 80% of infections occurring in areas that contain approximately one-third of the total population. Risk factors statistically associated with infection in a multivariate logistic regression model were living in the areas of high incidence, young age, and having an occupation including jhum cultivation and/or daily labor. Use of long lasting insecticide-treated bed nets was high (89.3%), but its use was not associated with decreased incidence of infection.Here we show that P. falciparum malaria continues to be hypoendemic in the Chittagong Hill Districts of Bangladesh, is highly seasonal, and is much more common in certain geographically limited hot spots and among certain occupations

Subclinical Plasmodium falciparum infections act as year-round reservoir for malaria in the hypoendemic Chittagong Hill districts of Bangladesh

Objectives: An analysis of the risk factors and seasonal and spatial distribution of individuals with subclinical malaria in hypoendemic Bangladesh was performed. Methods: From 2009 to 2012, active malaria surveillance without regard to symptoms was conducted on a random sample (n = 3971) and pregnant women (n = 589) during a cohort malaria study in a population of 24 000. Results: The overall subclinical Plasmodium falciparum malaria point prevalence was 1.0% (n = 35), but was 3.2% (n = 18) for pregnant women. The estimated incidence was 39.9 per 1000 person-years for the overall population. Unlike symptomatic malaria, with a marked seasonal pattern, subclinical infections did not show a seasonal increase during the rainy season. Sixty-nine percent of those with subclinical P. falciparum infections reported symptoms commonly associated with malaria compared to 18% without infection. Males, pregnant women, jhum cultivators, and those living closer to forests and at higher elevations had a higher prevalence of subclinical infection. Conclusions: Hypoendemic subclinical malaria infections were associated with a number of household and demographic factors, similar to symptomatic cases. Unlike clinical symptomatic malaria, which is highly seasonal, these actively detected infections were present year-round, made up the vast majority of infections at any given time, and likely acted as reservoirs for continued transmission

Quantification of host-mediated parasite clearance during blood-stage Plasmodium infection and anti-malarial drug treatment in mice

A major mechanism of host-mediated control of blood-stage Plasmodium infection is thought to be removal of parasitized red blood cells (pRBCs) from circulation by the spleen or phagocytic system. The rate of parasite removal is thought to be further increased by anti-malarial drug treatment, contributing to the effectiveness of drug therapy. It is difficult to directly compare pRBC removal rates in the presence and absence of treatment, since in the absence of treatment the removal rate of parasites is obscured by the extent of ongoing parasite proliferation. Here, we transfused a single generation of fluorescently-labelled Plasmodium berghei pRBCs into mice, and monitored both their disappearance from circulation, and their replication to produce the next generation of pRBCs. In conjunction with a new mathematical model, we directly estimated host removal of pRBCs during ongoing infection, and after drug treatment. In untreated mice, pRBCs were removed from circulation with a half-life of 15.1 h. Treatment with various doses of mefloquine/artesunate did not alter the pRBC removal rate, despite blocking parasite replication effectively. An exception was high dose artesunate, which doubled the rate of pRBC removal (half-life of 9.1 h). Phagocyte depletion using clodronate liposomes approximately halved the pRBC removal rate during untreated infection, indicating a role for phagocytes in clearance. We next assessed the importance of pRBC clearance for the decrease in the parasite multiplication rate after high dose artesunate treatment. High dose artesunate decreased parasite replication ∼46-fold compared with saline controls, with inhibition of replication contributing 23-fold of this, and increased pRBC clearance contributing only a further 2.0-fold. Thus, in our in vivo systems, drugs acted primarily by inhibiting parasite replication, with drug-induced increases in pRBC clearance making only minor contributions to overall drug effect