Ektacytometry Analysis of Post-splenectomy Red Blood Cell Properties Identifies Cell Membrane Stability Test as a Novel Biomarker of Membrane Health in Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is the most common form of hereditary chronic hemolytic anemia. It is caused by mutations in red blood cell (RBC) membrane and cytoskeletal proteins, which compromise membrane integrity, leading to vesiculation. Eventually, this leads to entrapment of poorly deformable spherocytes in the spleen. Splenectomy is a procedure often performed in HS. The clinical benefit results from removing the primary site of destruction, thereby improving RBC survival. But whether changes in RBC properties contribute to the clinical benefit of splenectomy is unknown. In this study we used ektacytometry to investigate the longitudinal effects of splenectomy on RBC properties in five well-characterized HS patients at four different time points and in a case-control cohort of 26 HS patients. Osmotic gradient ektacytometry showed that splenectomy resulted in improved intracellular viscosity (hydration state) whereas total surface area and surface-to-volume ratio remained essentially unchanged. The cell membrane stability test (CMST), which assesses the in vitro response to shear stress, showed that after splenectomy, HS RBCs had partly regained the ability to shed membrane, a property of healthy RBCs, which was confirmed in the case-control cohort. In particular the CMST holds promise as a novel biomarker in HS that reflects RBC membrane health and may be used to asses treatment response in HS.

Resistance of Plasmodium falciparum malaria to chloroquine is widespread in eastern Afghanistan.

After two decades of war and conflict in Afghanistan, the public-health system is in disarray and malaria has re-emerged as a major disease, with Plasmodium falciparum malaria becoming increasingly common. The limited healthcare services that are available are mainly delivered by non-governmental organizations in collaboration with the Ministry of Health. Although chloroquine (CQ) remains the official first-line treatment against P. falciparum malaria, there is little information on the severity or distribution of resistance to this drug in Afghanistan. In-vivo surveys, co-ordinated by the Malaria Reference Centre in Jalalabad, were therefore performed to determine the frequency and grades of CQ resistance in the three eastern provinces of Kunar, Nangarhar and Laghman. Of the 142 cases enrolled in the study, only 47 (33%) were sensitive. Most of the cases (55%) showed RI resistance but RII/RIII resistance was not uncommon (11%). The prevalence of resistance appeared similar in children and adults, in males and females, and in each of the three provinces investigated. Gametocyte carriage post-treatment was elevated in the resistant cases. As in neighbouring Pakistan, the resurgence of P. falciparum in Afghanistan is probably associated with the transmission and spread of chloroquine-resistant strains. The first-line therapy used against P. falciparum malaria must be changed in order to reverse this trend.

Daily iron supplementation is more effective than twice weekly iron supplementation in pregnant women in Pakistan in a randomized double-blind clinical trial.

In the context of limited effectiveness of iron supplementation programs, intermittent iron supplementation is currently under debate as a possible alternative strategy that may enhance the effectiveness of operational programs. This field-based trial assessed the outcome of twice weekly iron supplementation compared to daily in Pakistan. A double-blind, randomized, clinical trial was conducted in Northern Pakistan. Anemic pregnant women (n = 191) were assigned to receive daily (200 mg ferrous sulfate) or twice weekly (2 x 200 mg ferrous sulfate) iron supplementation. Hemoglobin was measured at baseline and at 4-wk intervals for up to 12 wk. Serum ferritin was measured at baseline and 8 or 12 wk. Analysis was by intention to treat. The two groups did not differ in age, parity, sociodemographic characteristics, hemoglobin or serum ferritin concentrations at baseline. Women who received iron daily had a greater rise in hemoglobin compared with women who received iron twice weekly (17.8 +/- 1.8 vs. 3.8 +/- 1.2 g/L, P < 0.001). The serum ferritin concentrations increased by 17.7 +/- 3.9 microgram/L (P < 0.001) in the daily supplemented group and did not change in the twice weekly group. Daily iron supplementation remained superior to twice weekly supplementation after controlling initial hemoglobin Z-scores and duration of treatment. The body mass index (BMI) modified the effect of daily versus twice weekly iron supplementation. For every unit increase in BMI, the difference between the two treatment groups was reduced by 0.0014 (final hemoglobin Z-score; P = 0.027). We recommend continuation of daily iron supplementation as opposed to intermittent iron supplementation in pregnant women in developing countries.

Snake bite in the Thar Desert.

Snake bite remains a common injury in endemic areas of Pakistan. A facility based observational study was undertaken to ascertain the incidence of snake bite, its mortality and the management in public health facilities located in the Thar Desert of Province of Sindh, Pakistan. A total of 771 cases of snake bite were reported to seven randomly selected health facilities during a one-year period. Most (75%) occurred during nighttime in summer, affecting primarily males (70%) who were bitten mostly on the lower extremities (83%). Five hundred and thirteen bites (66%) were identified as poisonous and 4 deaths were reported. Seventy two percent of patients reached the health facility within 24 hours and twenty percent within 6 hours and of being bitten. Snake bite remains an important problem in endemic areas of Pakistan. People do seek assistance at public health facilities, where appropriate treatment including anti snake venom is given.

Water-borne hepatitis E virus epidemic in Islamabad, Pakistan: a common source outbreak traced to the malfunction of a modern water treatment plant.

During December 1993 and in the first three months of 1994, an explosive water-borne epidemic of hepatitis E virus (HEV) occurred in two sectors of Islamabad, Pakistan. In a survey of a population of 36,705 individuals, a total of 3,827 cases of acute icteric hepatitis were recorded with an overall attack rate (AR) of 10.4%. The etiologic role of HEV in the epidemic was proven by demonstrating anti-HEV IgG and HEV IgM in the tested serum samples. The water-borne nature of the epidemic was suggested by a study of the case distribution according to water supply. Prior to the epidemic, there had been an operational breakdown in a water treatment plant that distributed water to the affected areas while transforming its purification system from slow sand to rapid sand filtration. The primary source of water for the plant was derived from a heavily contaminated stream. The highest AR (16.3%) was observed in the areas where the source of drinking water was exclusively from the purification plant, followed by ARs of 12.4% and 5.3% for those receiving 50% and 30% or less of their water supply from the treatment plant, respectively, while the lowest AR (1.8%) was observed in the neighboring areas that did not receive water from this source. The AR was significantly higher in the group 11-30 years of age (15.3%) as compared with children less than 11 years of age (1.4%) and also relative to the group greater than 30 years of age (10.5%). The AR among the 162 recorded pregnant females was 21.6%, which was higher than that found among nonpregnant females of child-bearing age (10.9%). All four reported adult deaths occurred among females in their third trimester of pregnancy with a case fatality rate of 11.4%, while the other four fatal cases were newborn infants of mothers with acute icteric hepatitis. Although the aggregation of cases within households was significantly related to family size, the temporal relationship between cases in households with two or more cases revealed that 83.7% of 1,463 presumed secondary cases occurred within one month of the first case in the same household, which is not suggestive of person-to-person transmission of disease. The termination of providing water from the source was effected, which was followed by an apparent decrease in cases.

Detection of anti-Leishmania antibodies in blood collected on filter paper by the direct agglutination test.

Human capillary blood drawn by finger pricking was absorbed on Whatman no. 4 filter paper and tested for anti-Leishmania antibodies using the direct agglutination test (DAT). Over 1400 filter paper samples were collected from L. infantum endemic areas in northern Pakistan and compared with 569 serum samples obtained from the same area. Eleven filter paper samples (0.8%) gave positive results (antibody titre > or = 1:3200), compared with 8 serum samples (1.4%); the difference was not statistically significant (P = 0.35). The filter paper eluates compared well with their homologous sera, and were strongly correlated with respect to antibody titre (r = 0.7, P < 0.0001).

Cutaneous leishmaniasis: iso-enzyme characterisation of Leishmania tropica.

In order to identify and characterise the organisms responsible for Cutaneous Leishmaniasis, parasites were isolated from active lesions, grown in-vitro cultures and identified by iso-enzyme characterisation. Thirteen isolates from different patients were typed as L. tropica. Seven of these isolates were from Afghan refugees encamped in the suburbs of Islamabad, 3 were from patients in Multan, 1 was from a patient from Azad Jammu and Kashmir and 1 was from Besham (Swat, NWFP). The study confirms the presence of anthroponotic Cutaneous Leishamaniasis caused by L. Tropica in Pakistan.

The role of dogs in the epidemiology of human visceral leishmaniasis in northern Pakistan.

Infantile visceral leishmaniasis (VL) was first reported from north Pakistan over 3 decades age in the remote valleys of the western Himalayas. These foci were reported as being completely devoid of domestic dogs. The later emergence of sporadic cases of infantile VL in the sub-Himalayan region of the country, where dogs are abundant, enabled us to investigate the prevalence of canine disease and study its relation with disease in humans. A serological survey in dogs by direct agglutination test (DAT) and enzyme-linked immunosorbent assay (ELISA) indicated that 18% (DAT) and 26.6% (ELISA) harboured anti-Leishmania antibodies, with older dogs showing higher prevalence; 10% of the infected dogs had no clinical signs of leishmaniasis. Deoxyribonucleic acid (DNA) probing by 32P-labelled Lmet 2 cDNA probe showed high sensitivity with aspirates obtained from the popliteal lymph nodes of dogs but not with skin snips. Parasites isolated from dogs in these foci were identified as L. infantum by isoenzyme characterization.

Leishmania infantum in the Himalayas.

Visceral leishmaniasis (VL) was first reported in Pakistan over 30 years ago from the remote north-eastern Himalayan region. Sporadic cases are now widely reported from the Northern Areas, part of North-West Frontier Province (NWFP) and Punjab Province, together with Azad Jammu and Kashmir (AJK). Two hundred and thirty-nine cases, mostly young children, have been reported in the last decade; 52% were under 2 years old, and 86% were aged less than 5 years; the male:female ratio was 3:1. In a cross sectional study of the population in 14 endemic villages, 1938 individuals of all ages were randomly skin-tested with leishmanin and 27.4% gave a positive result. The leishmanin response rate increased with age. In some areas, almost 70% of the adults over 40 years old were positive. Leishmanin positivity appeared to cluster among households (P = 0.0003), but clinical cases among households did not (P = 0.472). The leishmanin response pattern in the foci of Chilas and AJK reflected an endemic pattern of disease, whereas that seen in the Baltistan foci suggested interrupted transmission. Human sera collected in endemic villages were tested for anti-Leishmania antibodies, 580 by enzyme-linked immunosorbent assay and direct agglutination test (DAT), and another 1403, as blood samples on filter paper, were tested by DAT alone. Isolations of Leishmania were made from 15 patients, 11 from bone marrow, 3 from normal skin and one from spleen. The parasites were typed as Leishmania infantum zymodeme LON-49 (= MON-1).

Dracunculiasis: an approach to hasten worm expulsion.

Twenty patients with Guinea worm disease having a total of 29 emerging worms in a rural village of North West Frontier Province, were put on a 5-day course of antibiotics and antiinflammatory drugs and compared with age-matched control patients. The ulcer sites of emerging worms were kept continuously moist, and 18 worms were extracted from 14 patients during the above mentioned period. The average time taken for the entire worm to be expelled in these patients was considerably shorter (13.6 days) than in controls (89 days). Seven (39%) of these worms were expelled spontaneously whereas the remaining 11 (61%) were pulled out manually with relative ease. Fifteen (75%) patients in this study had a single emerging worm, three (15%) had two emerging worms and two (10%) patients had four or more emerging worms.

Sero-epidemiological survey of malaria by immunofluorescence in Pakistan (District Gujranwala-Punjab).

Indirect Immunofluorescence Antibody (IFA) Technique was employed, for field detection of malarial parasite. The technique involved application of homologous antigen slides of Plasmodium falciparum and P. vivax prepared in the field conditions. Serological study, simultaneously using P. falciparum and P. vivax antigens, brought out a clearer picture of distribution and role of the parasite species in the epidemiology of the disease in the area studied. The survey supported the contention of persistence of transmission of malaria in Gujranwala District.

Red Dracunculus medinensis.

Two instances of the emergence of a bright-red worm from humans in Pakistan are described. A 25-year-old man and a 26-year-old woman, unrelated and living in separate households, were observed in 1987 and 1988, respectively, to have emergent worms typical in size and location for Guinea worm disease, except for the color of the worms. The posterior end of 1 worm was recovered and appeared to be a female Dracunculus medinensis. Histologic sections provided insufficient information to determine the cause of the red color, although we could exclude blood as the cause.

Visceral leishmaniasis: a seroepidemiological study of 289 children from endemic foci in Azad Jammu and Kashmir by indirect fluorescent antibody technique.

Between January '85 and August '87, 22 cases of VL were seen at National Institute of Health, Islamabad. Three (14.6%) came from the previously known endemic region of Gilgit, 15 (68.1%) from different localities in Azad Jammu and Kashmir (AJ&K), and 4 (17.3%) from neighbouring foci in NWFP and Punjab. Mean age of the patients was 4.2 years, (Range, 10 months to 57 years) median 2.5 years and mode 2 years. High levels of Leishmania antibodies were detected by Indirect Immunofluorescent Antibody Technique (IFAT) in all cases. Leishmania were isolated from bone marrow aspirates of 2 patients and isoenzyme characterization performed in one of these, the organism was typed as Leishmania infantum sensu stricto. Sera from 289 children residing in 5 endemic localities in AJ&K was tested for Leishmania specific antibodies by IFAT and low levels of these antibodies were detected in 15.4% of the cases.