Vivax malaria and chloroquine resistance: a neglected disease as an emerging threat.

In Pakistan, Plasmodium vivax contributes to major malaria burden. In this case, a pregnant woman presented with P. vivax infection and which was not cleared by chloroquine, despite adequate treatment. This is probably the first confirmed case of chloroquine-resistant vivax from Pakistan, where severe malaria due to P. vivax is already an emerging problem.

The evolutionary history of Plasmodium vivax as inferred from mitochondrial genomes: parasite genetic diversity in the Americas.

Plasmodium vivax is the most prevalent human malaria parasite in the Americas. Previous studies have contrasted the genetic diversity of parasite populations in the Americas with those in Asia and Oceania, concluding that New World populations exhibit low genetic diversity consistent with a recent introduction. Here we used an expanded sample of complete mitochondrial genome sequences to investigate the diversity of P. vivax in the Americas as well as in other continental populations. We show that the diversity of P. vivax in the Americas is comparable to that in Asia and Oceania, and we identify several divergent clades circulating in South America that may have resulted from independent introductions. In particular, we show that several haplotypes sampled in Venezuela and northeastern Brazil belong to a clade that diverged from the other P. vivax lineages at least 30,000 years ago, albeit not necessarily in the Americas. We propose that, unlike in Asia where human migration increases local genetic diversity, the combined effects of the geographical structure and the low incidence of vivax malaria in the Americas has resulted in patterns of low local but high regional genetic diversity. This could explain previous views that P. vivax in the Americas has low genetic diversity because these were based on studies carried out in limited areas. Further elucidation of the complex geographical pattern of P. vivax variation will be important both for diversity assessments of genes encoding candidate vaccine antigens and in the formulation of control and surveillance measures aimed at malaria elimination.

Tumour necrosis factor, interleukin-6 and interleukin-10 are possibly involved in Plasmodium vivax-associated thrombocytopaenia in southern Pakistani population.

BACKGROUND: In Pakistan, Plasmodium vivax is endemic causing approximately 70% of the malaria cases. A number of haematological changes, especially thrombocytopaenia have been reported for P. vivax. Several host factors including cell-mediated immune cells, such as IL-1, IL-6 and IL-10 have been documented for P. vivax-induced thrombocytopaenia. However, study on correlation of cytokines and thrombocytopaenia in P. vivax, particularly in patients with severe signs and symptoms has not been reported from Pakistan. METHODS: A case control study to correlate TNF, IL-6 and IL-10 in healthy controls and thrombocytopaenic P. vivax-infected patients (both uncomplicated and complicated cases) from southern Pakistan was carried out during January 2009 to December 2011. One Hundred and eighty two patients presenting with microscopy-confirmed asexual P. vivax mono-infection and 100 healthy controls were enrolled in the study at Aga Khan University Hospital, Karachi. Enzyme-linked immunosorbent assay (ELISA) was performed for determination of TNF, IL-6 and IL-10 levels. RESULTS: Out of 182 cases, mild thrombocytopaenia (platelet count 100,000-150,000 mm3) was observed in ten (5.5%), moderate (50,000-100,000 mm3) in 93 (51.1%), and profound thrombocytopaenia (<50,000 mm3) was detected in 79 (43.4%) patients. IL-6 and IL-10 levels were found approximately three-fold higher in the mild cases compared to healthy controls. Two-fold increase in TNF and IL-10 (p < 0.0001) was observed in profound thrombocytopaenic when compared with moderate cases, while IL-6 was not found to be significantly elevated. CONCLUSION: Cytokines may have a possible role in P. vivax-induced thrombocytopaenia in Pakistani population. Findings from this study give first insight from Pakistan on the role of cytokines in P.vivax-associated thrombocytopaenia. However, further studies are required to understand the relevance of cytokines in manifestations of thrombocytopaenia in P. vivax malaria.

Genetic diversity of Plasmodium vivax clinical isolates from southern Pakistan using pvcsp and pvmsp1 genetic markers.

BACKGROUND: Plasmodium vivax is the prevalent malarial species accounting for 70% of malaria burden in Pakistan; however, there is no baseline data on the circulating genotypes. Studies have shown that polymorphic loci of gene encoding antigens pvcsp and pvmsp1 can be used reliably for conducting molecular epidemiological studies. Therefore, this study aimed to bridge the existing knowledge gap on population structure on P. vivax from Pakistan using these two polymorphic genes. METHODS: During the period January 2008 to May 2009, a total of 250 blood samples were collected from patients tested slide positive for P. vivax, at the Aga Khan University Hospital, Karachi, or its collection units located in Baluchistan and Sindh Province. Nested PCR/RFLP was performed, using pvcsp and pvmsp1 markers to detect the extent of genetic diversity in clinical isolates of P. vivax from southern Pakistan. RESULTS: A total of 227/250 (91%) isolates were included in the analysis while the remainder were excluded due to negative PCR outcome for P.vivax. Pvcsp analysis showed that both VK 210 (85.5%, 194/227) and VK 247 type (14.5%, 33/227) were found to be circulating in P. vivax isolates from southern Pakistan. A total of sixteen and eighty-seven genotypes of pvcsp and pvmsp-1 were detected respectively. CONCLUSION: This is the first report from southern Pakistan on characterization of P. vivax isolates confirming that extensively diverse pvcsp and pvmsp1 variants are present within this region. Results from this study provide valuable data on genetic diversity of P. vivax that will be helpful for further epidemiological studies.

Prevalence and molecular basis of glucose-6-phosphate dehydrogenase deficiency in Afghan populations: implications for treatment policy in the region.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PD), an x-linked inherited enzymopathy, is a barrier to malaria control because primaquine cannot be readily applied for radical cure in individuals with the condition. In endemic areas, including in Afghanistan, the G6PD status of vivax patients is not routinely determined so the drug is rarely, if ever, prescribed even though it is included as a recommended treatment in local, regional and global guidelines. This study assessed the prevalence and genotype of G6PD deficiency in Afghan populations and examined the need for routine G6PD testing as a malaria treatment and control tool. METHODS: A cross-sectional household survey was conducted using random sampling in five Afghan cities to determine the prevalence of G6PD deficiency in Afghan ethnic groups. Filter-paper blood spots were analysed for phenotypic G6PD deficiency using a fluorescent spot test. Molecular analysis was conducted to identify the genetic basis of the disorder. RESULTS: Overall, 45/1,436 (3.1%) people were G6PD deficient, 36/728 (5.0%) amongst males and 9/708 (1.3%) amongst females. Amongst males the prevalence was highest in the Pashtun ethnic group (10%, 26/260) while in Tajik males it was 8/250 (3.2%); in Hazara males it was 1/77 (1.3%) and in Uzbek males is was 0/125. Genetic testing in those with deficiency showed that all were of the Mediterranean type (Med-) characterized by a C-T change at codon 563 of the G6PD gene. CONCLUSION: Prevalence of G6PD deficiency in Afghanistan varies considerably by ethnic group and is predominantly of the Mediterranean type. G6PD deficient individuals are susceptible to potentially severe and life-threatening haemolysis after standard primaquine treatment. If the aim of increasing access to radical treatment of vivax is to be successful reliable G6PD testing needs to be made routinely available within the health system.

Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan.

BACKGROUND: Scarce data are available on Plasmodium falciparum anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of P. falciparum resistance associated polymorphisms in field isolates from southern Pakistan. METHODS: Blood samples from 244 patients with blood-slide confirmed P. falciparum mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt K76T), multi drug resistance (pfmdr1 N86Y), dihydrofolate reductase (pfdhfr A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (pfdhps A436S, G437A and E540K) genes and pfmdr1 gene copy numbers were determined using PCR based methods. RESULTS: The prevalence of pfcrt 76T and pfmdr1 86Y was 93% and 57%, respectively. The prevalence of pfdhfr double mutations 59R + 108N/51R + 108N was 92%. The pfdhfr triple mutation (51I, 59R, 108N) occurred in 3% of samples. The pfdhfr (51I, 59R, 108N) and pfdhps (437G, 540E) quintuple mutation was found in one isolate. Pfdhps 437G was observed in 51% and 540E in 1% of the isolates. One isolate had two pfmdr1 copies and carried the pfmdr1 86Y and pfcrt 76T alleles. CONCLUSIONS: The results indicate high prevalence of in vivo resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among P. falciparum in southern Pakistan.

Genetic diversity among Plasmodium falciparum field isolates in Pakistan measured with PCR genotyping of the merozoite surface protein 1 and 2.

BACKGROUND: The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2). METHODS: Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC. RESULTS: A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC). CONCLUSIONS: This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.

Plasmodium vivax Mimicking Morphologic Features of Plasmodium falciparum.

Plasmodium vivax (P. vivax) is the most common cause of malaria in Pakistan. Several cases of severe malaria due to P.vivax have been reported from Pakistan and India, however morphological characteristics of the parasite have been mainly ignored. We present two cases of P. vivax mono-infection, which were characterized by multiple infected red blood cells, similar to that seen in Plasmodium falciparum, as observed under microscopy. Both cases were confirmed as mono-infection of P.vivax on Giemsa stained thick and thin films, malaria rapid diagnostic test (RDT) and Polymerase Chain Reaction (PCR). Morphology on peripheral blood smear remains the gold standard for diagnosis of malaria and mimicking morphological features leads to misdiagnosis and mismanagement of patients.

Estimation of parasite load using Rapid diagnostic test ICT Now Malaria P.f/P.v in Plasmodium falciparum malaria.

Rapid tests such as ICT Malaria are an effective field tool in determining the presence of malarial parasites but do not provide an estimate of parasite load. We have evaluated the utility of ICT for providing semi-quantitative estimates of parasite load. Circulating parasite load in the blood of patients with malaria (n =54), were compared with the circulating HRP2 protein levels. Blood was serially diluted and analysed by a rapid diagnostic test (ICT(R) Now P.f/P.v) for assessment of endpoint PfHRP2 antigen titres. Significant correlation was observed between parasite load and PfHRP2 antigen titres (Spearman rank; rho = 0.821; p<0.001) with plasma dilutions > 1:16 corresponding to a parasite load of 0.1% parasitaemia. Variability in haematological parameters had no effect on the antibody titres obtained with the ICT test. Rapid semi-quantitative assessment of parasite load in conjunction with the Plasmodium speciation may provide a useful bedside and field aid in the diagnosis of malaria.

Hematological Profile and Gametocyte Carriage in Malaria Patients from Southern Pakistan.

Background Malarial infection is a major cause of concern, both worldwide and in Pakistan. Gametocytes are the sexual forms of the parasite that are essential for transmission. They fuse inside the mosquito to develop sporozoites. Gametocytes of the plasmodium parasites, which cause the infection, differentiate into male and female gametocytes. These gametocytes constitute the sexual stage of the malaria parasite and are essential in transmission of the disease from human to vector Anopheles. Gametocytes are affected by factors such as host immunity, drug treatment, reticulocytemia, anemia, low levels of asexual parasitemia and stress to the parasite. The aim of this study was to observe the hematological parameters, age and gametocyte carriage in an area of seasonal malaria transmission. Methods The study was conducted at Aga Khan University Hospital (AKUH) Laboratory over the period of one year and 294 patients with uncomplicated malaria were recruited. Patients infected with Plasmodium falciparum (P. falciparum) or Plasmodium vivax (P. vivax) malaria and no co-morbidities were included in the study. Results Gametocytemia was highest during the period of July to November, with P. vivax, 267 (90.8%), predominating compared to P. falciparum, 27 (9.2%). P. vivax gametocytes were observed from May to October and P. falciparum gametocytes were observed from July to December. Low hemoglobin in females and low platelet levels were observed. The mean platelet count was significantly lower in cases of P. vivax having gametocytes compared to P. falciparum with gametocytes. Higher parasitic index was associated with lower platelet count. The most significantly altered parameters were hemoglobin, hematocrit, white blood cell (WBC), and platelet count. Hemoglobin and platelets were significantly lower during the malaria season in study participants, both male and female. Conclusion In conclusion, infection with P. falciparum and P. vivax modulates significant changes in hematological parameters in populations living in malaria endemic regions. In the malaria season males were more frequently affected by malaria with thrombocytopenia. Gametocyte carriage remains unaffected by seasonal changes thus ensuring parasite transmission during the dry season.

Diagnostic Value of Gauze Filtration Technique: A Comparison with Conventional Methods in a Diagnostic Laboratory in Pakistan.

Background Intestinal parasites cause significant morbidity and impact human development with an enormous global burden. Diagnosis of intestinal parasites by conventional methods has several limitations. The gauze filtration technique is a relatively simple method that has been shown to identify intestinal parasites with a high sensitivity and specificity. The aim of this study was to determine the diagnostic value of this technique as compared to more conventional methods in a large acclaimed laboratory within Pakistan. Methods A total of 50 stool samples collected for routine diagnostic workup from patients age between 2-70 years were collected from the parasitology section of the Aga Khan University Hospital Clinical Laboratory. A direct wet mount, sedimentation technique, and gauze filtration technique were performed on all of the stool samples, and the sensitivity, specificity, negative predictive value, and positive predictive value were analyzed. Results It was observed that the number of organisms observed by gauze filtration as compared to direct wet mount and sedimentation technique was higher for B. hominis, G. lamblia cysts and trophozoites, and I. bütschlii. Also, the detection rate was significantly higher for B. hominis and G. lamblia cysts using the gauze filtration technique. The sensitivity and specificity of the gauze filtration technique were found to be 95.8% and 100%, respectively. Conclusion There is a significantly better stool sample parasite detection rate using the gauze filtration technique as compared to the conventional sedimentation techniques. The utility of the gauze filtration technique seems economically and technically feasible for diagnostic laboratories in resource-limited settings.