Genetic structure of Plasmodium falciparum field isolates in eastern and north-eastern India.

BACKGROUND: Molecular techniques have facilitated the studies on genetic diversity of Plasmodium species particularly from field isolates collected directly from patients. The msp-1 and msp-2 are highly polymorphic markers and the large allelic polymorphism has been reported in the block 2 of the msp-1 gene and the central repetitive domain (block3) of the msp-2 gene. Families differing in nucleotide sequences and in number of repetitive sequences (length variation) were used for genotyping purposes. As limited reports are available on the genetic diversity existing among Plasmodium falciparum population of India, this report evaluates the extent of genetic diversity in the field isolates of P. falciparum in eastern and north-eastern regions of India. METHODS: A study was designed to assess the diversity of msp-1 and msp-2 among the field isolates from India using allele specific nested PCR assays and sequence analysis. Field isolates were collected from five sites distributed in three states namely, Assam, West Bengal and Orissa. RESULTS: P. falciparum isolates of the study sites are highly diverse in respect of length as well as sequence motifs with prevalence of all the reported allelic families of msp-1 and msp-2. Prevalence of identical allelic composition as well as high level of sequence identity of alleles suggest a considerable amount of gene flow between the P. falciparum populations of different states. A comparatively higher proportion of multiclonal isolates as well as multiplicity of infection (MOI) was observed among isolates of highly malarious districts Karbi Anglong (Assam) and Sundergarh (Orissa). In all the five sites, R033 family of msp-1 was observed to be monomorphic with an allele size of 150/160 bp. The observed 80-90% sequence identity of Indian isolates with data of other regions suggests that Indian P. falciparum population is a mixture of different strains. CONCLUSION: The present study shows that the field isolates of eastern and north-eastern regions of India are highly diverse in respect of msp-1 (block 2) and msp-2 (central repeat region, block 3). As expected Indian isolates present a picture of diversity closer to southeast Asia, Papua New Guinea and Latin American countries, regions with low to meso-endemicity of malaria in comparison to African regions of hyper- to holo-endemicity.

Evaluation of bifenthrin treated mosquito nets against anopheline and culicine mosquitoes.

BACKGROUND AND OBJECTIVES: The main rural malaria vector Anopheles culicifacies has developed resistance to dichloro diphenyl trichloroethane (DDT), hexachloro cyclo hexane (HCH) and malathion in the state of Haryana in northern India. An alternative synthetic pyrethroid insecticide bifenthrin was therefore evaluated on mosquito nets against anopheline and culicine mosquitoes, in two villages Jagdishpur and Garh Mirakpur of Community Health Center (CHC) Badhkhalsa in district Sonipat, Haryana state. METHODS: Two formulations of bifenthrin, suspension concentrate (SC) and micro-emulsion (ME) were compared with micro-capsule suspension (CS) of lambdacyhalothrin. The impact of three doses of bifenthrin (10, 25 and 50 mg/m(2)) impregnated on mosquito nets was compared with lambdacyhalothrin (25 mg/m(2)) and untreated control. Quality assessment of treatment on treated nets was carried out by residue analysis and the persistence of the insecticide on nets was determined by contact bioassays. Efficacy of treated nets on mosquito density was assessed by calculating mosquito entry rate, immediate mortality, delayed mortality and excito-repellency to the insecticides. RESULTS: In susceptibility tests An. culicifacies was susceptible to bifenthrin (0.1% test papers) and to lambdacyhalothrin (0.05% test papers). Bioassays on treated nets against A. culicifacies recorded 100 per cent mortality up to tenth fortnight for all the doses of impregnation with bifenthrin (SC and ME) and lambdacyhalothrin (CS). Ring-net bioassays against An. culicifacies showed median knock-down time between 3.1 to 11.4 min. Behavioural indices were also studied for anopheline and culicine mosquitoes. The reduction in entry rates of anopheline and culicine mosquitoes into the rooms with treated nets compared to control indicated good efficacy with all the formulations and doses of the insecticides. INTERPRETAION AND CONCLUSION: Indoor (immediate) mortality of mosquitoes with bifenthrin ME formulation was relatively lower compared to SC fomulation of bifenthrin and based on delayed mortility and continued susceptibility in bioassays, bifenthrin ME at the rate of 10 mg/m(2) dose was found suitable for the impregnation of mosquito nets for phase III trial.

Study of the pituitary-thyroid functions at high altitude in man.

The alterations in serum levels of T3, T4, TSH and TBG, TSH response to 100 mug iv TRH, and urinary excretion of T3 and T4 were studied in 8 healthy men at sea level (SL), on days 1, 2, 4, 8 and 16 after arrival by air at high altitude (3,700 m, HA), and during days 5 to 7 after their return to SL. No significant alterations in serum levels of TSH and TBG or TSH response to TRH were observed during exposure to HA or on return to SL. There was, however, an acute elevation in both serum total T3 and T4. Serum total T3 from a mean basal+/-SE value of 128+/-13 ng/dl increased to 320+/-18 on day 1 and remained significantly elevated at 225+/-48 up to day 8 after arrival at high altitude. Similarly serum total T4 increased from basal level of 9+/-0.92 mug/dl to 15.2+/-1.2 and remained elevated till day 16 and it was 11+/-1.19 mug/dl during days 5 to 7 after return to SL. The urinary excretion of both T3 and T4 was decreased. These changes perhaps were the result of complex physiologic adjustments on acute exposure to high altitude, like shrinkage of the T3 and T4 distribution pools, altered binding capacities of thyroid hormones binding proteins, and a reduction in clearance of thyroid hormones from the plasma compartment; and were probably not suggestive of an enhanced thyroid activity. Their actual significance in high altitude adaptation in man is not clearly understood.

Utilization of different quantities of fat at high altitude.

Investigations on utilization of fats have been carried out at altitudes of 3,500, 3,800, and 4,700 m, respectively, on 27, 7, and 12 soldiers staying at these altitudes for over 4 months in tentage accommodation. At 3,500 m the fat utilization was 95.9, 96.2, and 96.6% on intakes of 128, 168, and 198 g fat/day. At 3,800 m on maximum fat intake of 364 g/day, the fecal fat content was 11.48 g and percentage digestibility 96.9. At 4,700 m the digestibility was 97.5% on a fat intake of 232 g/day. Urine examination did not reveal ketone bodies on any of these fat intakes. Neither was there any incidence of constipation nor diarrhea. While 364 g of fat/day could not be fully consumed, digestibilty was not affected up to an intake of 324 g/day though, at this intake there was feeling of thirst at night. Thus, digestibility and utilization of dietary fats up to 324 g at 3,800 m and 232 g at 4,700 m are not distrubed.

Three case reports of the metabolic and electroencephalographic changes during advanced Buddhist meditation techniques.

To examine the extent to which advanced meditative practices might alter body metabolism and the electroencephalogram (EEG), we investigated three Tibetan Buddhist monks living in the Rumtek monastery in Sikkim, India. In a study carried out in February 1988, we found that during the practice of several different meditative practices, resting metabolism (VO2) could be both raised (up to 61%) and lowered (down to 64%). The reduction from rest is the largest ever reported. On the EEG, marked asymmetry in alpha and beta activity between the hemispheres and increased beta activity were present. From these three case reports, we conclude that advanced meditative practices may yield different alterations in metabolism (there are also forms of meditation that increase metabolism) and that the decreases in metabolism can be striking.

Ethnic variations in psychological performance under altitude stress.

Sea-level performance of 120 subjects equally drawn from Gorkha, Madrasi, and Rajput groups was compared with that at an altitude of 4,000 m after 10 months' stay. Alterations in concentration, eye-hand coordination, anxiety, depression, and social interaction were recorded to ascertain ethnic differences in withstanding the altitude stress. Results showed a uniform fall in the scores for eye-hand coordination and social interaction at altitude for the three ethnic groups. However, the effects on concentration, anxiety, and depression were differential for these groups indicating a better toleration of altitude stress by Gorkhas. Climatic similarity between the region of natural habitat of Gorkhas and altitude environment has been suggested to be responsible for their better performance over the other two groups.

Changes in orthostatic tolerance in man at an altitude of 3500 meters.

Orthostatic tolerance was measured in 20 lowlander Indian soldiers (sojourners) by recording responses of heart rate (HR), blood pressure (BP) and mean skin temperature (Tsk) to 70 degrees head-up passive tilt, initially at Delhi (260 m altitude) and thereafter at 3500 m at weekly intervals for 3 weeks. For comparison, observations were also made once on 10 acclimatized lowlanders (AL) and 10 high-altitude natives (HAN) at the same altitude. Among sojourners, the percentage of subjects showing orthostatic intolerance (OI) during tilt increased at high altitude in the first and second weeks; six subjects fainted in the first week as compared to one who fainted at sea level (SL). There was no incidence of fainting among AL or HAN and the percentage of subjects showing OI was very small. The magnitude of cardio-acceleration to tilt was higher in sojourners at high altitude (HA) and relatively less in AL and HAN. The systolic BP during tilt showed no change at SL or during the first and second weeks of stay at altitude in sojourners, but in third week showed a mild rise and was comparable to the responses of AL and HAN. The diastolic BP during tilt always showed a rise, although it was of a higher magnitude during the second and third weeks in sojourners as well as in AL and HAN. There was a fall in Tsk during tilt, which was of a lesser magnitude at HA. It has been concluded that there is a reduced orthostatic tolerance at higher altitude during the first week, due to hypocapnia, after which it is improved as a result of relative sympathetic hyperactivity and adaptation of the vasomotor centre to reduced PaCO2 level.

Electroencephalography in naval divers.

EEG studies were conducted on 98 naval divers to ascertain the incidence of abnormal paroxysmal activity in them and to correlate it with their performance in diving. Nine of the 98 showed EEG abnormality in the form of a buildup of high-voltage, low-frequency activity after hyperventilation. None of them showed sporadic bursts of sharp waves from any region on intermittent photic stimulation. A 5-year follow-up did not show any association between the EEG response and the diver's professional capabilities. Nine divers were also examined after a 30-min simulated exposure at 2.5 ATA breathing air. This simulated dive had no effect on the existing EEG pattern. It has been concluded that a routine EEG examination is not useful in selection of divers.

Effect of rewarming at various water bath temperatures in experimental frostbite.

Studies have been conducted on 72 rats to determine the most suitable temperature at which rapid rewarming should be done as an immediate treatment for frostbite. Animals were put in a harness containing arrangements for warming the body. Their hind limbs were left out of the harness. They were then exposed to -15 degrees +/- 1 degrees C in a deep freeze for 60 min, during which paw temperature was recorded every 5 min. After this, the animals were taken out, the left hind limb was rapidly rewarmed in a water bath maintained at 35 degrees, 37 degrees, 39 degrees, 41 degrees, 43 degrees, or 45 degrees C for different batches and the right hind limb was left free for slow rewarming at room temperature (27 degrees -29 degrees C). The severity of cold injury in the two limbs was compared. The paw temperature showed a drop on cold exposure, followed by a rapid rise and then a second fall. The degree of injury was related to the duration of exposure after the rise in the paw temperature. The rapid rewarming was effective only at water bath temperature of 37 degrees-39 degrees C and was harmful at 45 degrees C. This shows that rewarming at about body temperature is most effective as immediate treatment for frostbite.

Potassium losses in sweat under heat stress.

Six healthy, heat-acclimatized subjects were exposed to different hot and humid environments in a climatic chamber and sodium, potassium, and chloride concentrations in their sweat, urine, and blood were determined. The concentration of potassium in sweat was found to be considerably higher than that in the plasma, whereas that of sodium and chloride was very much lower. The concentration of potassium in urine was also 8-12 times higher than that in the plasma as compared to 0.5 to 1.5 times higher for sodium and chloride. The total daily computed losses of potassium in sweat and urine, of a person working in severe heat in the tropics, can be about 116 mEq as against a dietary intake of 97 mEq/d, thereby resulting in negative potassium balance. The potassium depletion in sweat, even in acclimatized Indians, is thus heavy and is likely to play an important role in the causation of heat-illness.

Cold-induced vasodilatation response at different water bath temperatures in monkeys.

The response of cold-induced vasodilatation (CIVD) at different water bath temperatures was studied in 20 monkeys (3.5 kg) in a conscious state in a thermoneutral room. The animals were controlled by seating in a monkey chair, and the right hind limb up to 7.5 cm from the heel was immersed in a water bath for 60 min. Four series of experiments were conducted at water bath temperatures of 0 degrees, 4 degrees, 8 degrees, and 12 degrees C, respectively, at weekly intervals and the skin temperatures were measured from three sites in the foot. Marked CIVD response was noted from the dorsum and, to a lesser extent, from the sole of the foot, but no response was seen from the tip of the middle toe at 0 degrees, 4 degrees, and 8 degrees C water bath temperatures. The pattern of CIVD response at 4 degrees C was identical to that of 0 degrees C, but the response at 8 degrees C was poor and was absent at 12 degrees C. Three patterns of CIVD--such as hunting, proportional control, and slow, steady, and continuous rewarming--was observed. However, 15% of the animals did not exhibit any CIVD. The observations show that the CIVD response of monkeys is remarkably similar to that of man.

Cold-induced vasodilatation and peripheral blood flow under local cold stress in man at altitude.

The cold-induced vasodilatation (CIVD) response was studied on 17 lowlanders (20-30 years) at Delhi, using a water bath maintained at 4 degrees+/-0.2 degreesC. The temperatures were measured at the tip of the index finger, centre of the palm, over a prominent wrist vein, and orally. Then the subjects were air-lifted to an altitude of 3500 m, where the measurements were repeated at weekly intervals for a period of 3 weeks. After this, they were flown back and retested. For comparison, the study at altitude was done on 10 acclimatised lowlanders and on 10 high-altitude natives. The peripheral blood flow under a local cold stress was calculated. Three types of CIVD responses--viz. typical hunting, proportional control, and continuous rewarmin--were observed. At altitude, the pattern was more or less the same but there was a significant reduction in the response. The responses of the natives were more or less identical to those of the lowlanders at sea level, and responses of the acclimatized lowlanders were better than the fresh inductees, but much less than the natives. The changes in peripheral blood flow was in accordance with the CIVD response.

Effect of altitude acclimatization on thermoregulation efficiency of man.

A study has been conducted on 20 sojourners (SJs) at a high altitude (HA) of 3500 m to evaluate the changes in thermoregulation efficiency (ThE) during acclimatization for 3 weeks, by observing the rate and pattern of rewarming of palm skin temperature (Tsk) after dipping the hand in water of 10 +/- 1 degrees C for 2 min (Tromp's water bath test). The ThE of the SJs was compared to that of 10 acclimatized lowlanders (ALs) staying at the same altitude for about 1 year and 10 high-altitude natives (HANs). In SJ, at sea level (SL), the effect of seasonal variation and ambient temperature on ThE was also studied. The initial Tsk of the palm (29.9 degrees C) was lower at altitude as compared to SL value (32.2 degrees C) in SJs inspite of similar ambient temperature (21 +/- 1 degrees C). There was a prolongation of rewarming time in all the subjects during stay at altitude. The rewarming curve showed a shift towards the right, indicating slower rewarming. The rewarming response of ALs and SJs was comparable at altitude. In HANs, the rewarming at altitude was faster and resembled those of SJs at SL. The rewarming improved in the season following cold months, and at higher ambient temperature in the same season, on the plains. It may be concluded that thermoregulation efficiency of man deteriorates at high altitude, and the peripheral vascular responses to local cold stress do not reach the level attained by natives even after 1 year of acclimatization. General cold acclimatization improves the rewarming response.

Electrolyte changes at 3500 m in males with and without high-altitude pulmonary edema.

Ten normal healthy male subjects between 20-30 years of age were initially examined at Delhi (200 m) and thereafter air-lifted to an altitude of 3,500 m. Excretion of sodium, potassium and chloride in urine and their plasma level were determined at sea level (SL) and daily at high altitude (HA) for 4 d. At HA, four subjects developed high-altitude pulmonary edema (HAPE), four remained normal, and two suffered from acute mountain sickness. The results on normals and HAPE are presented. There was increased excretion of potassium at HA in both groups resulting in reduction of plasma level. The sodium and chloride excretion was also increased in normals at HA irrespective of urine volume. In HAPE cases, the sodium and chloride excretion was related to urine output. With the retention of fluid, the excretion of these ions in urine was diminished without a parallel change in plasma levels.

Haematologic changes in rabbits during acclimatisation, deacclimatisation, and reinduction to hypoxia.

Haematologic changes were studied in 16 albino rabbits during acclimatisation, deacclimatisation, and reinduction to hypobaric hypoxia. Of these, four animals died during reinduction. Haemoglobin, haematocrit, and red blood cell count attained maximum values by the 15th day during acclimatisation and by the 10th day during reinduction. Reticulocyte count reached maximum level by the fifth day, both during acclimatisation and reinduction. All these parameters returned to initial control levels by the 10th day of deacclimatisation. The erythrocytes became macrocytic during acclimatisation and remained so during deacclimatisation and reinduction. Blood volume increased on acclimatisation, returned to control values during deacclimatisation, and remained low at this level on reinduction. Red cell mass increased both during acclimatisation and reinduction and returned to control levels on deacclimatisation. Plasma volume did not change on acclimatisation and deacclimatisation, but was reduced on reinduction. The erythrocyte life span and erythrocyte glutamic oxaloacetic transaminase (EGOT) decreased during acclimatisation and reinduction. There was no change in RBC osmotic fragility, indicating no change in the integrity of the red cell wall. The deaths during reinduction were possibly due to haemoconcentration and increased blood viscosity as a result of reduction in plasma volume. It has been concluded that reinduction to hypoxia after a phase of deacclimatisation imposes a more severe stress than experienced during acclimatisation.

Changes in EEG pattern during acclimatization to high altitude (3500 m) in man.

This study was conducted on 30 healthy soldiers to observe EEG changes during acclimatization to high altitude. Of these subjects, 10 were lowlanders (LL), 10 were acclimatized low landers (AL), and 10 10 were high-altitude natives (HAN). The EEG was recorded by the 10-20 international system of electrode placement in LL at sea level (SL) during rest and hyperventilation (HV). Then they were air-lifted to an altitude of 3500 m where periodic recordings of EEG were made for 4 weeks. For comparison, the altitude recordings were also made in AL and HAN. The quantitative analysis of EEG (occipital) was done to determine alpha index (AI) and average amplitude. The mean AI was 25.5% at SL in LL, which changed to 45.7%, 15.8%, 28.0%, 30.3%, and 33.2% on days 2, 7, 14, 21, and 28, respectively. The average amplitude was 17.3 microV at SL, which changed to 23.3 microV, 11.8 microV, 16.2 microV, 17.3 microV, and 19.8 microV on days 2, 7, 14, 21, and 28, respectively. In two subjects, spiking was seen along with the desynchronized pattern of EEG on days 7 and 14. The AI and average amplitude of AL and HAN were significantly more than those of LL. The magnitude of buildup during HV was greater at altitude. These results indicate that there is cerebral cortical depression in the initial phase of induction which changes to cortical desyncronization in the latter part of the first week of induction. During acclimatization, there is gradual buildup of EEG waves, as observed in AL and HAN.

Responses of the autonomic nervous system during acclimatization tp high altitude in man.

A study has been conducted on 20 sojourners, between the ages of 20-30 years, to evaluate responses of the autonomic nervous system during acclimatization to high altitude. The responses measured consisted of heart rate (HR), blood pressure (BP), oral temperature (Tor), mean skin temperature (Tsk), cold pressor response (CPR), orthostatic tolerance to tilt, and urinary catecholamines. The subjects were tested initially at Delhi (altitude 260 m) and thereafter, on acute induction to an altitude of 3500 m periodically for 3 weeks. For comparison, the same responses were studied on 10 acclimatized lowlanders (AL) who had been staying at the same altitude for more than a year and on 10 high-altitude natives (HAN). The studies showed a rise in HR, BP, Tor, and urinary catecholamines, and a fall in Tsk, CPR, and orthostatic tolerance immediately on arrival at HA, indicating a relative hyperactivity of the sympathetic system. After a stay of 1 week, there was a gradual recovery in all the responses, though sympathetic hyperactivity was still maintained throughout the 3 weeks of stay. In AL also there was a preponderance of sympathetic activity, though of relatively lesser magnitude than that seen in sojourners. In HAN, on the other hand, there was a relative parasympathetic predomination. It has been concluded that in lowlanders it takes more than a year of stay at altitude for complete recovery of autonomic balance.

Glucose tolerance of lowlanders during prolonged stay at high altitude and among high altitude natives.

The fasting blood sugar level and glucose tolerance were investigated in seven amle lowlanders at sea level, during their stay at an altitude of 4,000 m at intervals of 2 weeks, 10, 15, 20, and 24 months and again on return to sea level during the first week and after 1 month. For comparison, the glucose tolerance of six male Ladakhis (natives of high altitude area) was also determined at altitude. The fasting blood sugar among lowlanders increased to 136.0 plus or minus 4.39 mg per 100 ml during 10 months of stay at altitude followed with a gradual decrease to a value of 76.4 plus or minus 3.8 at the end of 24 months. On return to sea level, the blood glucose showed a tendency to increase. Ladakhis had a lower blood sugar level at altitude (86.4 plus or minus 7.28) as compared with lowlanders at sea level (92.6 plus or minus 2.29). The glucose tolerance curves of lowlanders ran paralell to each other at altitude and at sea level. However, the peak of the glucose tolerance curve shifted towards the left during the 20th and 24th months of stay at altitude and at sea level. However, the peak of the glucose tolerance curve shifted towards the left during the 20th and 24th months of stay at altitude and on return to sea level. The tolerance curve of Ladakhis was similar to that of lowlanders at altitude, but showed a sharper blood sugar decline rate.

Body fluid compartments in rabbits on exposure to acute hypobaric hypoxia.

Body fluid compartments were studied in rabbits divided into three groups--control, exposed to acute hypoxia, and exposed to hypoxia after treatment with 2 mg frusemide intramuscularly. Total body water, extracellular body water, and plasma space were determined using the triple radiotracer technique. Total body water decreased insignificantly with no change in extracellular body space on exposure to hypoxia. Plasma volume and blood volume showed a significant decrease with a significant increase in haematocrit. In rabbits pretreated with frusemide, total body water, extracellular body water, plasma volume, blood volume, and interstitial fluid space decreased significantly on hypoxic exposure. This study suggested hypohydration on acute hypoxic exposure with a loss of intracellular water, while pretreatment with frusemide resulted in further hypohydration with a loss from both intracellular and extracellular compartments. The results have been discussed in relation to suggested use of diuretics on induction to high altitude.

Studies on heat output from the hand of frostbite subjects.

We studied 12 subjects, who had suffered first- to third-degree frostbite at high altitude during winter, at Delhi, India. At normal sea level pressure there (PB 740 mm Hg) and in a decompression chamber at a simulated altitude of 4085 m, the studies were at both 26 degrees C and 6.8 degrees C. A group of control (non-frostbite) subjects of comparable age were also studied for their heat output at 26 degrees C, PB 740 mm Hg. Heat output from the hands of a group of mountaineers from the sea level was also studied at 2121 m at 25 degrees C and 4485 m at 7 degrees C. The results indicated that the frostbite subjects had a significantly higher heat ouput at PB 740 mm Hg and 26 degrees C than the non-frostbite subjects. When the former were tested at sea level (PB 740 mm Hg) at 6.8 degrees C, the hand heat output showed a marked and significant decrease. On testing them at a simulated altitude of 4085 m at 26 degrees C and at 6.8 degrees C, a very highly significant reduction in hand heat output was observed compared to their initial value at sea level (740 mm Hg) and 26 degrees C. Their hand heat output also showed a very highly significant decrease compared to mountaineers at 4485 m and 7 degrees C.