Population genetics and drug resistance markers: an essential for malaria surveillance in Pakistan

Plasmodium (P.) vivax is the prevalent malarial species accounting for 70% of malaria cases in Pakistan. However, baseline epidemiological data on P. vivax population structure and drug resistance are lacking from Pakistan. For population structure studies, molecular genetic markers, circumsporozoite protein (csp) and merozoite surface protein-1 (msp-1) are considered useful as these play an important role in P. vivax survival under immune and environmental pressure. Furthermore, these genes have also been identified as suitable candidates for vaccine development. While efforts for effective vaccine are underway, anti-malarial agents remain the mainstay for control. Evidence of resistance against commonly used anti-malarial agents, particularly Sulphadoxine-Pyrimethamine (SP) is threatening to make this form of control defunct. Therefore, studies on drug resistance are necessary so that anti-malarial treatment strategies can be structured and implemented accordingly by the Malaria Control Program, Pakistan. This review aims to provide information on genetic markers of P. vivax population structure and drug resistance and comment on their usefulness in molecular surveillance and control

Plant Root Hair in Tap Water: A Potential Cause for Diagnostic Confusion

Plant root hairs are commonly found artifacts in parasitology specimens and may be confused with helminthes by an untrained eye. We report a case of brain tuberculoma where the tissue sample was contaminated with root hair derived from tap water; the presence of this root hair, which mimicked a larva, led to diagnostic confusion. Therefore, tap water should be considered a source of root hair and vegetable matter

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

Prevalence of drug resistance associated mutations in plasmodium vivax against sulphadoxine-pyrimethamine in southern Pakistan

Background: In Pakistan, Plasmodium vivax and Plasmodium falciparum co-exist and usage of sulphadoxine-pyrimethamine (SP) against P. falciparum exposes P. vivax to the drug leading to generation of resistant alleles. The main aim of this study was to investigate frequency distribution of drug resistance associated mutations in pvdhfr, pvdhps genes and provide baseline molecular epidemiological data on SP-associated resistance in P. vivax from southern Pakistan.Methods: From January 2008 to May 2009, a total of 150 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 using pvdhfr and pvdhps specific primers was performed for all samples.91.3% (137/150) of the samples were tested PCR positive of which 87.3% (131/137) were successfully sequenced. Sample sequencing data was analysed and compared against wild type reference sequences.Results: In dhfr, mutations were observed at codons F57L, S58R and S117N/T. Novel non-synonymous mutations were observed at codon positions N50I, G114R and E119K while a synonymous mutation was observed at codon position 69Y. In dhps, mutations were observed at codon position A383G and A553G while novel non-synonymous mutations were observed at codon positions S373T, E380K, P384L, N389T, V392D, T393P, D459A, M601I, A651D and A661V.Conclusion: This is the first report from southern Pakistan on SP resistance in clinical isolates of P. vivax. Results from this study confirm that diverse drug resistant alleles are circulating within this region

Hydatid cyst disease of the thorax

Hydatid disease is a frequently encountered entity in endemic areas and constitutes a serious health problem. The lung is the second most commonly affected organ after the liver. In three of four cases of pulmonary hydatid cyst, the cyst is solitary. The early phase after primary infection is always asymptomatic. Primary symptoms in patients with pulmonary cystic echinococcosis include cough, fever, and chest pain. The clinical and radiological manifestations for the hydatid cyst in the lung depend on the integrity of the cyst. Bronchial fistulization is an important event in the evolution of the cyst. In cases of dubious orientation after the initial study, ultrasonography, conventional tomography, and magnetic resonance imaging prove helpful in delineating the disease accurately. For a specific serologic diagnosis, theimmunoglobulin G enzyme-linked immunosorbent assay and immunoelectrophoresis are preferred. Despite the fficacy of new chemotherapeutic regiments, the mainstay of treatment for thoracic hydatidosis remains essentially surgical. In general chemotherapy is used as a complement to surgical treatment to avoid recurrence of the disease. Regardless of the surgical methods adopted, the removal of the parasite intoto, prevention of its dissemination, maximum preservation of pulmonary function, and the immediate obliteration of the remaining cavity are the imperative basis for effective therapy. The most frequent post-surgical complications are pleural infection and prolonged air leakage. The operative morbidity and recurrence rates have been reported with variation. Education of the population in endemic areas regarding the disease transm ission cycle and institution of appropriate prophylaxis are essential steps to eradicate the disease completely

Entamoeba species associated with chronic diarrhoea in Pakistan

We determined the prevalence of Entamoeba (E.) histolytica, E. dispar and E. moshkovskii in patients with chronic diarrhoea associated with abdominal pain or discomfort mimicking irritable bowel syndrome. Stool samples were collected from 161 patients with chronic diarrhoea and from 157 healthy controls. Stool microscopy with modified trichrome stain, culture and polymerase chain reaction (PCR) for Entamoeba spp. differentiation was performed. Microscopy demonstrated Entamoeba cysts in 44% (57/129) of patients with diarrhoea compared to 29% (44/151) of controls (P=0·009). In patients with diarrhoea, PCR for E. histolytica was positive in 9% (11/129) (P=0·008), E. dispar in 19% (24/129) (P=0·117) and E. moshkovskii in 19% (24/129) (P\u3c0·001). E. histolytica and E. moshkovskii were significantly associated with diarrhoea while E. dispar was found equally in both groups

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 malaria reported during summer and winter at a tertiary care hospital in Karachi, Pakistan

This study was designed to determine the prevalence and type of malaria cases that presented throughout the year 2014 in a tertiary care hospital in Karachi, Pakistan. A total of 1099 cases, (377 females, 722 males) were reported. Plasmodium vivax (P. vivax) was discovered in 93.7% cases compared to 6.3% Plasmodium falciparum (P. falciparum). Based on the highest and lowest weather temperatures, in summer (June, July and August) and in winter (December, January and February) were differentiated. The number of cases were greater during summer months compared to winter. Interestingly, the ratio of P. falciparum to P. vivax during winter was greater compared to summer. Finally, there was a strong correlation between increasing humidity and number of malaria cases. These findings show that even though the incidence of malaria is higher in summer, malaria cases are still reported in winter. Furthermore, the probability of finding P. falciparum (which causes cerebral malaria ) is higher in winter

Irritable bowel syndrome: is it associated with genotypes of Blastocystis hominis.

Blastocystis hominis is the most common intestinal parasite in humans. An extensive genetic variability has been described recently in B. hominis isolates. The aim of this study was to analyze genotypes of B. hominis isolates obtained from the healthy individuals and patients with irritable bowel syndrome-diarrhea (IBS-D). The patients with IBS-D were enrolled from gastroenterology outpatient department at the Aga Khan University Hospital. History and physical examination was done. Stool microscopy, culture, and polymerase chain reaction for B. hominis genotyping were carried out. The study included 158 patients with IBS-D, mean age 41 +/- 15, age range 16-83 years, and male/female ratio of 109:49. One hundred fifty-seven (49.8%) were taken as healthy control. The dominant B. hominis genotypes were genotype 1 in 87 (65%) and type 3 in 49 (37%). In IBS-D, genotype 1 was present in 75 (86%; P \u3c 0.001) compared to 12 (14%) in controls while type 3 was present in 23 (47%) compared to 26 (53%) in controls (P \u3c 0.001), respectively. Infection with single genotype of B. hominis was present in 70 (73%) with IBS-D and in 26 (27%) in control group while with multiple genotypes in 25 (64%) in IBS-D and 14 (36%) in control group (P = 0.30), respectively. Majority of our patients had typeable B. hominis infection. The genotype of B. hominis in IBS-D was type 1 while in control genotype 3 was predominant