Molecular surveillance of drug resistance: Plasmodium falciparum artemisinin resistance single nucleotide polymorphisms in Kelch protein propeller (K13) domain from Southern Pakistan

Background: K13 propeller (k13) polymorphism are useful molecular markers for tracking the emergence and spread of artemisinin resistance in Plasmodium falciparum. Polymorphisms are reported from Cambodia with rapid invasion of the population and almost near fixation in south East Asia. The study describes single nucleotide polymorphisms in Kelch protein propeller domain of P. falciparum associated with artemisinin resistance from Southern Pakistan.Methods: Two hundred and forty-nine samples were collected from patients with microscopy confirmed P. falciparum malaria attending Aga Khan University Hospital during September 2015-April 2018. DNA was isolated using the whole blood protocol for the QIAmp DNA Blood Kit. The k13 propeller gene (k13) was amplified using nested PCR. Double-strand sequencing of PCR products was performed using Sanger sequencing methodology. Sequences were analysed with MEGA 6 and Bio edit software to identify specific SNP combinations.Results: All isolates analysed for k13 propeller allele were observed as wild-type in samples collected post implementation of ACT in Pakistan. C580Y, A675V, Y493H and R539T variants associated with reduced susceptibility to artemisinin-based combination therapy (ACT) were not found. Low frequency of M476I and C469Y polymorphisms was found, which is significantly associated with artemisinin resistance.Conclusion: Low frequencies of both nonsynonymous and synonymous polymorphisms were observed in P. falciparum isolates circulating in Southern Pakistan. The absence of known molecular markers of artemisinin resistance in this region is favourable for anti-malarial efficacy of ACT. Surveillance of anti-malarial drug resistance to detect its emergence and spread need to be strengthened in Pakistan

Neurological complications in patients with plasmodium vivax malaria from Karachi, Pakistan

Background: Malaria remains an endemic disease in Pakistan with an estimated healthcare burden of 1.6 million cases annually, with Plasmodium vivax accounting for 67% of reported cases. P. vivax is the most common species causing malaria outside of Africa, with approximately 13.8 million reported cases worldwide. Method: We report a series of P. vivax cases with cerebral involvement that presented at Aga Khan University Hospital, Karachi, Pakistan. Results: The majority of the patients presented with high-grade fever accompanied by projectile vomiting and abnormal behaviour, seizures, shock and unconsciousness. Seven of 801 patients with P. vivax monoinfection presented or developed cerebral complications. P. vivax infections were diagnosed based on peripheral smears and rapid diagnostic testing. Conclusion: P. vivax infection can lead to severe complications, although not with the frequency of Plasmodium falciparum infection. Current cases highlight an increasing trend of cerebral complications caused by P. vivax

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

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

Neurological involvement associated with plasmodium vivax malaria from Pakistan

Plasmodium vivax is the most common specie causing malaria outside Africa with approximately 13.8 million reported cases worldwide. We report case of P. vivax infection with cerebral involvement. A nine year old boy presented with high grade fever accompanied by projectile vomiting and abnormal behavior later he developed seizures, shock, and unconsciousness. P. vivax monoinfection was diagnosed based on peripheral smears and PCR. After antimalarial therapy, patient made full recovery. Current case highlights increasing trend of cerebral complications caused by P. vivax

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

Frequency of G6PD mediterranean in individuals with and without malaria in southern Pakistan

Background: Pakistan has an estimated annual burden of 1.5 million malaria cases. The current situation calls for an efective malaria control and eradication programme in this country. Currently, primaquine is an attractive option for eliminating reservoirs of Plasmodium vivax hypnozoites and killing gametocytes of Plasmodium falciparum. However, this drug causes haemolysis in individuals who are glucose-6-phosphate (G6PD) defcient. It is important to map G6PD defciency and malaria distribution in Pakistan to design an efective malaria eradication regimen. Frequency of G6PD defciency (G6PDd) in malaria patients has not been reported from Pakistan in any meaningful way. The purpose of this study was to evaluate the frequency of G6PD c.563C\u3eT (G6PD Mediterranean) in male individuals with and without falciparum malaria.Methods: Two hundred and ten archived DNA samples from males (110 from falciparum malaria patients and 100 from healthy individuals) were utilized in this study. Healthy blood donors were selected based on stringent predefned criteria. Patients were confrmed for malaria parasites on microscopy and or immune chromatographic assay detecting P. falciparum histidine-rich protein 2. Parasitaemia was also computed. DNA samples were tested for G6PD c.563C\u3eT mutation through PCR–RFLP according to the previously defned protocol and its allelic frequency was computed.Results: G6PD c.563C\u3eT was observed in four of 110 patients with falciparum malaria and in two of 100 healthy donors. Mean (± SD) haemoglobin, median (IQR) platelet and median (IQR) parasite count in G6PD-defcient malariapatients were 8.9 ± 0.9 g/dL, 124 × 109/L (IQR 32, 171) and 57,920/μL of blood (IQR 12,920, 540,000) respectively.Conclusions: Cumulative allelic frequency for G6PD 563c.C\u3eT was 0.0285 detected in 6 of 210 X-chromosomes in Southern Pakistan. Frequency for this G6PD allele was 0.0364 in malaria-patients and 0.0200 in healthy individuals. Large studies including females are needed to elucidate the true burden of G6PDd in malaria-endemic areas. The information will enable local health policy makers to design efective strategies for eliminating malaria form this region

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

Perception of pathology as a future career choice among medical students from Karachi, Pakistan: experience from a private medical school.

To determine the perception of pathology as a future career choice among medical students of a private medical school from Karachi, Pakistan. A descriptive cross-sectional study was conducted at the Aga Khan University, Karachi, Pakistan. A total of 201 students participated in this study. All Students were approached randomly to participate. A total of 201 students participant survey forms were evaluated in this study. The overall satisfaction level with pathology was observed in 61.8% ofthe students. Majority of the students understood subspecialties which were a part of clinical medicine. Over half of the students thought pathology as a specialty should be highlighted in a more integrated manner (59.2%) with a minority favouring a separate pathology rotation (11.9%). In conclusion, this study indicates that majority of students have a positive approach towards the field of pathology and favour incorporating it in an integrative way into the medical school curriculum

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

Implementing parasite genotyping into national surveillance frameworks: Feedback from control programmes and researchers in the Asia-pacific region

The Asia-Pacific region faces formidable challenges in achieving malaria elimination by the proposed target in 2030. Molecular surveillance of Plasmodium parasites can provide important information on malaria transmission and adaptation, which can inform national malaria control programmes (NMCPs) in decision-making processes. In November 2019 a parasite genotyping workshop was held in Jakarta, Indonesia, to review molecular approaches for parasite surveillance and explore ways in which these tools can be integrated into public health systems and inform policy. The meeting was attended by 70 participants from 8 malaria-endemic countries and partners of the Asia Pacific Malaria Elimination Network. The participants acknowledged the utility of multiple use cases for parasite genotyping including: quantifying the prevalence of drug resistant parasites, predicting risks of treatment failure, identifying major routes and reservoirs of infection, monitoring imported malaria and its contribution to local transmission, characterizing the origins and dynamics of malaria outbreaks, and estimating the frequency of Plasmodium vivax relapses. However, the priority of each use case varies with different endemic settings. Although a one-size-fits-all approach to molecular surveillance is unlikely to be applicable across the Asia-Pacific region, consensus on the spectrum of added-value activities will help support data sharing across national boundaries. Knowledge exchange is needed to establish local expertise in different laboratory-based methodologies and bioinformatics processes. Collaborative research involving local and international teams will help maximize the impact of analytical outputs on the operational needs of NMCPs. Research is also needed to explore the cost-effectiveness of genetic epidemiology for different use cases to help to leverage funding for wide-scale implementation. Engagement between NMCPs and local researchers will be critical throughout this process