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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.

Background Various factors determine the outcome of Plasmodium falciparum infection such as parasite load, sequestration, adhesion molecules, and immune mediators. P. falciparum merozoite surface protein-1 ( msp-1 ) and msp-2 genotypes were also found associated with severe disease. We investigated the association between msp-1 and msp-2 genotypes in patients with uncomplicated malaria (UM) and severe malaria (SM). Methods Twenty-two malaria patients with microscopy-confirmed P. falciparum infection and eight healthy endemic controls were selected for analysis. Nested polymerase chain reaction (PCR) was used to identify P. falciparum genotypes. The plasma concentration of cytokines [tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interferon-gamma (IFN-γ)] and chemokines [chemokine (C-X-C motif) ligand 9 (CXCL9) and CXCL10] were evaluated using enzyme-linked immunosorbent assay (ELISA). Results TNF-α levels were significantly higher in both UM (389 pg/mL, p = 0.020) and SM (771 pg/mL, p = 0.004) compared with healthy controls, while they were greater in SM ( p = 0.012) as compared to UM. CXCL9 levels were significantly raised in SM as compared to UM and negative controls (NCs). CXCL10 levels were raised in UM (550 pg/mL, p = 0.001) and SM (1480 pg/mL, p = 0.01) as compared with NCs. Increased levels of IL-6 were found in patients carrying the FC27 allelic type of msp-2 . A higher prevalence of MAD 20 and K1 msp-1 alleles was observed in the SM group compared to UM. Conclusion Overall, a greater prevalence of MAD 20 alleles and increased serum TNF-α and CXCL9 levels were associated with severe outcome in malaria. Understanding the diversity of malaria genotypes is important for predicting disease-related outcomes of P. falciparum infection in endemic areas.

Introduction An association of Helicobacter pylori and common protozoal parasites in patients with abdominal discomfort and chronic diarrhoea is unclear and may be pathological. Materials and methods One hundred and sixty-one patients with diarrhoea were compared to 114 age and sex matched controls. Stool samples were examined by microscopy and DNA extracted for PCR with specific primers for H. pylori and protozoal parasites Blastocystis sp., Entamoeba sp. (Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii) and Giardia duodenalis (G. duodenalis). Results There was a marked difference in the presence of parasites between patients and controls: no parasite 42/75%, one parasite 42/15%, two or more parasites 16/10%, respectively (p 0.001). Patients with diarrhoea were more likely to be infected with Blastocystis sp (p 0.001), E. histolytica (p = 0.027) and E moshkovskii (p = 0.003). There was no difference in the frequency of H. pylori (p = 0.528), G duodenalis (p = 0.697) or E dispar (p = 0.425). Thirty-three patients and 27 controls had H. pylori infection. Of these, 22 patients and 6 controls were infected with Blastocystis sp (p = 0.001), 6 patients and no controls were infected with E. histolytica (p = 0.02), whilst 7 patents and 9 controls were infected with E dispar (p = 0.292). Conclusion In this population, diarrhoea is linked to infection with Blastocystis sp, E. histolytica and E moshkoviskii. In H. pylori infection, diarrhoea is linked to Blastocystis sp and E. histolytica infection. These associations may be linked pathogenically.

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.

Background Tuberculoma and cerebral infarctions are serious complications of central nervous system (CNS) tuberculosis. However, there are no studies comparing prognostic value of tuberculoma and infarcts alone and in patients diagnosed with CNS tuberculosis. Objective The objective of this study was to identify frequency and prognostic value of tuberculoma and cerebral infarcts in a large sample of CNS tuberculosis patients. Methods Retrospective chart review of patients diagnosed with CNS tuberculosis in a tertiary care hospital in Pakistan over 10-year period was carried out. Results There were 404 patients included in this study (mean age of 43 years). There were 209 (52%) men and 195 (48%) women. Tuberculoma were present in 202 subjects (50%) while infarcts were present in 25% patients. 147 (36%) had tuberculous meningitis (TBM) without tuberculoma or infarction on CT or MRI, 158 (39%) had TBM with intracranial tuberculomas, 60 (15%) had TBM with cerebral infarction while 39 (10%) had TBM with both tuberculoma and infarction. At discharge, 249 patients (62%) were either normal (Modified Rankin Score (MRS)=0) or mild to moderately disabled (MRS=1–3) while 82 patients (20%) had severe disability (MRS=4–5). 73 (18%) patients died (MRS=6) during hospitalisation. Using logistic regression analysis, significant predictors of poor outcome included old age, high TBM grading, presence of infarction and presence of hydrocephalus. Conclusions Tuberculomas were present in 50% of patients, while infarcts were present in 25%. Old age, TBM grading, presence of infarction and hydrocephalus were all predictors of poor outcome.

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.

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with point mutations in the P. falciparum CQ resistance transporter gene ( pfcrt ). Previous studies have shown 4–5 independent origins for CQ resistant pfcrt alleles globally, two in South America, one each in Southeast Asia, Papua New Guinea (PNG) and Philippines. In Asia, at least two different alleles corresponding to amino acids 72–76 (CVIET and SVMNT) have been found. The CVIET allele originated in Southeast Asia and then spread to Asia and Africa as well. The SVMNT allele, originating from PNG, has been found in India. This study was undertaken to investigate the genetic background of the CQ resistant pfcrt haplotypes in Pakistan. We genotyped microsatellite markers surrounding the pfcrt gene (six different markers at −12.3, −4.8, −1, 1.5, 3.9, 18.8 kb) in 114 clinical isolates of P. falciparum collected from different regions in Pakistan. Microsatellite analysis showed a significant reduction in genetic variation among the mutant SVMNT pfcrt alleles when compared to wild type alleles. The predominant SVMNT haplotype found in this study shared the same microsatellite haplotype found in both PNG and India. Two isolates with CVIET haplotypes showed similar microsatellite background to those found in Africa and Asia. In conclusion, this study suggests that CQ resistant SVMNT haplotypes in India and Pakistan have a common ancestral origin similar to that of Papua New Guinean isolates.

3 Abstract: A total of five-hundred and fifty (550) day-old Cobb-500 broiler chicks were used for a period of six weeks to study the effect of different stocking densities and feeding regime on the performance of broiler chicken. The treatments under stocking densities were D1 (8 bird/m ), D2 (10 bird/m ), D3 (12 bird/m ) and 2 2 2 D4 (14 bird/m ). The form of feeds under feeding regime was F1 (mash), F2 (crumble) and F3 (pellet) feeds. 2 The study revealed that the lower stocking density D1 consumed significantly (p

SUMMARYWe 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) (PE. histolytica and E. moshkovskii were significantly associated with diarrhoea while E. dispar was found equally in both groups.

Giardia lamblia and Cryptosporidium parvum are both waterborne pathogens associated with diarrhoea in developing countries. In a recent study based at the Aga Khan University in Karachi, 334 adults aged 16-83 years (178 patients with chronic diarrhoea and 156 diarrhoea-free volunteers who acted as controls) were checked for infection with these parasites, using stool microscopy and/or PCR. Overall, 21 (6.3%) and 29 (8.7%) of the subjects were found positive for G. lamblia by microscopy and PCR, respectively, while the corresponding values for C. parvum were 13 (3.9%) and 14 (4.2%). Although, compared with the diarrhoea-free controls, the patients with diarrhoea were not significantly more likely to be found infected with Giardia, either by microscopy [15 (8.4%) v. six (3.8%); P=0.085] or PCR [19 (10.7%) v. 10 (6.4%); P=0.167], they were significantly more likely to be found infected with C. parvum, both by microscopy [11 (6.2%) v. two (1.3%); P=0.024] and by PCR [12 (6.7%) v. two (1.3%); P=0.014]. The 19 patients found PCR-positive for Giardia comprised 10 (67%) of the 15 found smear-positive for the same parasite but only nine (5%) of the 163 found smear-negative (k=0.545; P0.001). Similarly, the 12 patients found PCR-positive for Cryptosporidium comprised all 11 (100%) patients found smear-positive for the same parasite but only one (0.6%) of the 167 found smear-negative (k=0.954; P0.001). Although C. parvum was associated with chronic diarrhoea in the present study, the carriage of G. lamblia often appeared asymptomatic.

Studies have suggested a possible role for Blastocystis hominis and Dientamoeba fragilis in the etiology of irritable bowel syndrome (IBS). We studied the prevalence of B. hominis and D. fragilis in patients with IBS-diarrhea (IBS-D). Three hundred and thirty patients were enrolled, 171 (52%) with IBS-D and 159 (48%) were controls, respectively. Stool microscopy, culture, and polymerase chain reaction (PCR) for B. hominis and D. fragilis were done. B. hominis was positive by stool microscopy in 49% (83/171) of IBS compared to 24% (27/159) in control (p < 0.001). B. hominis culture was positive in 53% (90/171) in IBS compared to 16% (25/159) in control (p < 0.001). B. hominis PCR was positive in 44% (75/171) in IBS compared to 21% (33/159) in control (p < 0.001). D. fragilis microscopy was positive in 3.5% (6/171) in IBS-D compared to 0.6% (1/159) in control (p = 0.123). D. fragilis culture was positive in 4% (7/171) in IBS compared to 1.3% (2/159) in control (p = 0.176). D. fragilis PCR was positive in 4% (6/171) in IBS-D compared to 0% (0/159) in control (p = 0.030). B. hominis is common, while D. fragilis was less prevalent in our patients with IBS-D. B. hominis and D. fragilis culture had a better yield compared to stool microscopy and PCR.

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; p0.001) with plasma dilutions1:16 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.

Background. Fomites are objects that can become colonized and serve as vectors in the transmission of pathogenic microorganisms. Literature examining the knowledge of healthcare personnel about this method of spread of infection is lacking. We conducted a study to assess the knowledge, attitude, and practices of healthcare personnel across different areas of patient care regarding the spread of infections at a tertiary care hospital in Karachi, Pakistan.Methods. A descriptive, cross-sectional study was conducted among healthcare personnel using a self-administered questionnaire. The questionnaire contained sections pertaining to demographic details and knowledge, attitude, and practices regarding fomites and their role in the transmission of pathogens.Results. Three hundred and fifty-three participants completed the questionnaire: 168 were male and 185 were female. Laboratory coats, stethoscopes, and bedside curtains were most frequently identified as fomites by the participants. Medical students had significantly lower mean scores in the knowledge and attitude sections than consultant physicians, resident physicians, and nurses. Nurses scored higher than consultant physicians, resident physicians, and medical students regarding practices that minimize fomite-borne spread of infections. 95% of the participants scored above 50% on the knowledge component of the questionnaire, but only 32.3% scored above 50% in the practices section.Conclusions. Our results show a large gap between the knowledge about fomites acting as vectors in the spread of pathogens and practices done to minimize this spread. Possessing adequate knowledge is ineffectual until and unless it is translated into the proper application of infection control practices. Incorporating awareness sessions and exercises into curricula are a reasonable way to raise awareness regarding this subject.

Primary amoebic meningoencephalitis (PAM) is a rare but fatal infection caused by Naegleria fowleri. The infection is acquired by deep nasal irrigation with infected water. Patients present with signs and symptoms similar to pneumococcal meningitis, leading to delayed diagnosis and treatment and hence high mortality. We conducted a case–control study comparing culture proven cases of PAM with pneumococcal meningitis presenting to our center between April 2008 and September 2014. Only patients with blood and/or cerebrospinal fluid cultures positive for Streptococcus pneumoniae during the same time period were included for comparison. There were 19 cases of PAM and pneumococcal meningitis, each. When comparing PAM with pneumococcal meningitis, patients with PAM were more likely to be male (89.5 vs. 36.8 %), younger (mean age: 30 vs. 59 years), present with seizures (42.1 vs. 5.3 %). Both groups of patients presented with similar vital signs and there were no remarkable differences on physical examinations, Glasgow Coma Scale scores, laboratory and radiological investigations and cerebrospinal fluid parameters. PAM was also more likely to present if the city’s average maximum temperature was higher in the previous week (mean: 34.6 vs. 30 °C). There was history of fresh water contact in only one patient. On multivariate analysis, PAM was more likely if patients presented when the city’s average maximum temperature was high, being young males. PAM and pneumococcal meningitis remain virtually indistinguishable; however, these predictive features should be validated in a prospective study and may lead to a viable algorithm for early management of these patients.

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.

A new experimental model was developed in hamsters for amoebic abscess caused by Entamoeba histolytica. E. histolytica trophozoites were cultured in a liquid axenic medium, and then injected intradermally into the cheek pouch of the Syrian golden hamster, Mesocricetus auratus. Inoculation consistently resulted in abscess formation at the site in 20 of 22 (91%) study animals. The amoebic nature of the abscesses was confirmed by light microscopy and histopathologic examination. Abscess formation was maximal at day 12 post-inoculation. Potential applications of this simple and reliable model include further elucidation of the pathogenesis of invasive amoebiasis, studies of the host response to amoebae, and in vivo evaluation of chemotherapeutic agents that show in vitro efficacy against E. histolytica.

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.

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 (

BackgroundCytokine-mediated endothelial activation pathway is a known mechanism of pathogenesis employed by Plasmodium falciparum to induce severe disease symptoms in human host. Though considered benign, complicated cases of Plasmodium vivax are being reported worldwide and from Pakistan. It has been hypothesized that P.vivax utilizes similar mechanism of pathogenesis, as that of P.falciparum for manifestations of severe malaria. Therefore, the main objective of this study was to characterize the role of cytokines and endothelial activation markers in complicated Plasmodium vivax isolates from Pakistan.Methods and principle findingsA case control study using plasma samples from well-characterized groups suffering from P.vivax infection including uncomplicated cases (n=100), complicated cases (n=82) and healthy controls (n=100) were investigated. Base line levels of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Intercellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule-1(VCAM-1) and E-selectin were measured by ELISA. Correlation of cytokines and endothelial activation markers was done using Spearman's correlation analysis. Furthermore, significance of these biomarkers as indicators of disease severity was also analyzed. The results showed that TNF-α, IL-10, ICAM-1and VCAM-1 were 3-fold, 3.7 fold and 2 fold increased between uncomplicated and complicated cases. Comparison of healthy controls with uncomplicated cases showed no significant difference in TNF-α concentrations while IL-6, IL-10, ICAM-1, VCAM-1 and E-selectin were found to be elevated respectively. In addition, significant positive correlation was observed between TNF-α and IL-10/ ICAM-1, IL-6 and IL-10, ICAM-1 and VCAM-1.A Receiver operating curve (ROC) was generated which showed that TNF-α, IL-10, ICAM-1 and VCAM-1 were the best individual predictors of complicated P.vivax malaria.ConclusionThe results suggest that though endothelial adhesion molecules are inducible by pro-inflammatory cytokine TNF-α, however, cytokine-mediated endothelial activation pathway is not clearly demonstrated as a mechanism of pathogenesis in complicated P.vivax malaria cases from 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.

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.

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.

Although Plasmodium vivax usually causes benign uncomplicated malaria, it can occasionally result in severe disease with life-threatening, end-organ involvement generally seen with falciparum malaria. We report a case of cerebral malaria caused by P. vivax and review the literature on this subject.

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.

We report a case of dust mite carriage in a 56-year-old gentleman. Dust mites eggs and larvae were found in a stool sample which was taken for a routine clinical examination. He was completely asymptomatic with no history of rash, airway disease or other allergic manifestations associated with dust mites. We noticed that the oval structure of mite eggs resembled helminth eggs and therefore may be misidentified during routine clinical analysis. As the patient was otherwise healthy, it was concluded that no rigorous antiparasitic therapy was necessary to eliminate dust mites from his system.

Parasitic diseases are a major public health problem in the tropical and sub tropical countries including the subcontinent region. We aimed to assess methods of Parasitology education in medical schools of Karachi Pakistan.Ten medical schools in Karachi, Pakistan were sent a structured questionnaire collecting information on different aspects of Parasitology education. The collected data was analyzed using SPSS version 14.0.The response rate of this study was 90%. Majority of the schools in Karachi, Pakistan (78%) taught Parasitology concurrently with Microbiology, Pathology, Pharmacology and Forensic medicine in third and fourth year of undergraduate training. More than 20 hours were spent on teaching through didactic lectures (56%), interactive lectures (22%), problem based learning (PBL) (22%), clinical cases (11%) and small group discussions (89%). A Clinical Microbiologist or Parasitologist taught Parasitology by using transparencies, handouts and/or computer aids. Variation in education methods existed mainly in the private medical schools.Medical curricula were meeting the European standards for teaching of Parasitology. However, there is a need for revision and modification in the curricula owing to the high burden of parasitic diseases in the subcontinent region.

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.

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 < 0.001) compared to 12 (14%) in controls while type 3 was present in 23 (47%) compared to 26 (53%) in controls (P < 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.

Background Intestinal parasitic infections are endemic worldwide and have been described as constituting the greatest single worldwide cause of illness and disease. Poverty, illiteracy, poor hygiene, lack of access to potable water and hot and humid tropical climate are the factors associated with intestinal parasitic infections. The study aimed to estimate prevalence and identify factors associated with intestinal parasitic infections among 1 to 5 years old children residing in an urban slum of Karachi Pakistan. Methods and Principal Findings A cross sectional survey was conducted from February to June 2006 in Ghosia Colony Gulshan Town Karachi, Pakistan. A simple random sample of 350 children aged 1–5 years was collected. The study used structured pre-tested questionnaire, anthropometric tools and stool tests to obtain epidemiological and disease data. Data were analyzed using appropriate descriptive, univariate and multivariable logistic regression methods. The mean age of participants was 2.8 years and 53% were male. The proportions of wasted, stunted and underweight children were 10.4%, 58.9% and 32.7% respectively. The prevalence of Intestinal parasitic infections was estimated to be 52.8% (95% CI: 46.1; 59.4). Giardia lamblia was the most common parasite followed by Ascaris lumbricoides, Blastocystis hominis and Hymenolepis nana. About 43% children were infected with single parasite and 10% with multiple parasites. Age {Adjusted Odds Ratio (aOR) = 1.5; 95% CI: 1.1; 1.9}, living in rented households (aOR = 2.0; 95% CI: 1.0; 3.9) and history of excessive crying (aOR = 1.9; 95% CI: 1.0; 3.4) were significantly associated with intestinal parasitic infections. Conclusions Intestinal parasites are highly prevalent in this setting and poverty was implicated as an important risk factor for infection. Effective poverty reduction programmes and promotion of deworming could reduce intestinal parasite carriage. There is a need for mass scale campaigns to create awareness about health and hygiene.

Two individuals presented to the Aga Khan University Opthalmology service with foreign-body sensation, pain and redness in one of their eyes. Slit-lamp biomicroscopy revealed tiny larvae crawling around the conjunctival sac. They were mechanically removed under topical anaesthesia and preserved for light microscopy and photography. Comprehensive liaison was established with the Australian Centre for International Agriculture Research for identification of these larvae. Their morphology characterized them as members of fruit-fly, Oestridae family. At least one of them was positively identified as first instar larva of Oestrus ovis. This report describes the first instance of such infections in Southern Pakistan, as concluded after a Medline search.

Summary Hyper-reactive malarial splenomegaly (HMS) is diagnosed by the presence of massive splenomegaly, raised IgM and antimalarial antibodies and a response to antimalarial therapy. Although malaria is endemic to Pakistan, HMS is uncommon. We report on HMS in a patient with massive splenomegaly, positive Plasmodium falciparum polymerase chain reaction but normal immunoglobulin M antibody levels. The investigations were not consistent with any other diagnosis. HMS is also briefly reviewed.

The original version of this article unfortunately contained a mistake. In Table 2 we calculated the percentages presented for the stool microscopy and culture of B. hominis row wise rather than vertically in the column. As this was aimed to determine the distribution of B.hominis in IBS-D versus control group, we should have calculated the percentage column wise. However, percentages calculated row wise will not change the P value when calculated column wise. There was an error on our part that percentages presented for the stool microscopy and culture of B. hominis in the Table 2 should have been reported column wise. Please see amended Table 2.

Summary Objectives A comparison of clinical and laboratory features, diagnostic methods, drug treatment, and outcomes for patients hospitalized with malaria by Plasmodium species. Methods Records of 521 patients hospitalized during the four and half-year study period were analyzed. Results Infections were caused by Plasmodium vivax (51.8%), Plasmodium falciparum (46.5%), P. vivax plus P. falciparum (1.3%), and Plasmodium malariae (0.4%). Vomiting (odds ratio (OR)=1.86, p = 0.001) and abdominal pain (OR=1.60, p =0.024) occurred more frequently in patients infected with P. falciparum compared to P. vivax ; this was also the case for hepatomegaly, splenomegaly and jaundice. Low hemoglobin levels were common but were significantly lower with P. falciparum , and creatinine levels were significantly higher with P. falciparum . Treatment regimens consisted of single drug therapy (61.5%), appropriate combination therapy (15.8%), and inappropriate combination therapy (22.7%). Antimalarials given alone included chloroquine (38.7%), quinine (19%) and doxycycline (1.5%). The overall mortality was 1.7% ( n =9) and nearly 56% of patients developed disease complications, most commonly thrombocytopenia (36.4%), anemia (23.4%), and thrombocytopenia plus anemia (32.7%). Conclusions Despite resistance, chloroquine was prescribed in patients with malaria requiring hospitalization. We found a high proportion of single antimalarial drug use as well as inappropriate combination therapy (22.7%), and inadequate use of primaquine terminal prophylaxis. Physicians need to be acquainted with malaria treatment guidelines in an endemic zone.