Virulence potential of Group A streptococci isolated from throat cultures of children from north India.

BACKGROUND & OBJECTIVES: Rheumatic fever (RF)/rheumatic heart disease (RHD) caused by Group A streptococcus (GAS) are more prevalent in north India as compared to the western world, where invasive diseases are common. This could be due to variation in the virulence of GAS in different geographic locations. Hence, we studied the virulence potential of GAS isolated from the throat of children from north India. METHODS: Fifty GAS isolated consecutively, from children with mild pharyngitis (20), severe pharyngitis (24) and asymptomatic pharyngeal carriers (6), were characterized by emm typing and opacity factor (OF). Adherence and internalization of GAS in HEp-2 cells and opsonophagocytosis in convalescent serum samples were studied. RESULTS: Twenty emm types, six sequence types, and one non-typeable GAS were circulating in the community. emm type 74, 11, 68, StI129 and NS292 were most prevalent. Twenty seven (54%) GAS isolates were OF negative. Sixty five per cent of the most prevalent emm types were OF negative indicating their rheumatogenic potential. Adhesion of GAS ranged from 0.1 to 100 per cent. Forty eight per cent of GAS were highly adherent. Invasion of GAS in HEp-2 cells ranged between 0 to 30 per cent. Only 20 per cent isolates exhibited highest invasion. GAS were opsonophagocytosed with highly divergent efficiency ranging from 0 to 91.7 per cent. Nineteen GAS were not opsonophagocytosed and 15 multiplied during the assay. Isolates of the same emm type also varied in their virulence potential. INTERPRETATION & CONCLUSIONS: GAS isolates from the throat of children from north India belonged to several emm types, majority were OF negative, excellent adherents but poor invaders. This explains why throat infections in these children tend to lead to ARF/RHD rather than invasive diseases. A few isolates exhibiting high invasion efficiency indicate that GAS throat cultures can also lead to invasive diseases.

Group A Streptococcus virulence factors genes in north India & their association with emm type in pharyngitis.

BACKGROUND & OBJECTIVES: Group A streptococcal (GAS) pharyngitis, especially among children, leads to high prevalence of rheumatic fever (RF)/rheumatic heart disease (RHD) in India, as compared to the western world where invasive diseases are common. GAS encodes numerous virulence factors that cause diseases by exhibiting extraordinary biological diversity. Hence, we studied the virulence factors genes of GAS isolated from the throat of children with pharyngitis and also asymptomatic carriers. METHODS: Fifty GAS isolates cultured from throats of north Indian children aged 5-15 yr with mild pharyngitis (20), severe pharyngitis (24) and asymptomatic pharyngeal carriers (6), during 2000-2003 along with reference M1 strain were emm typed and characterized for virulence factors genes by PCR. The presence of virulence factors was also checked for their association with emm type in pharyngitis. RESULTS: Twenty emm types, six sequence types, and one non-typeable strain were found circulating in north India. The five most prevalent types were emm 74 (12%), 11 & StI129 (8% each) and emm 68 and NS292 (6% each). The spe B gene was found to be significantly higher (P=0.0007) in opacity factor (OF) negative isolates. emm 3, 11, 77, 86, 87, 109 and StI129 showed maximum virulence factors genes. INTERPRETATION & CONCLUSIONS: GAS isolates collected from throats of children from north India possess highly virulent antigens. This study also supports concept of isolate-associated virulence rather than type relatedness.

Epidemiology of group A streptococcal pharyngitis & impetigo: a cross-sectional & follow up study in a rural community of northern India.

BACKGROUND & OBJECTIVE: Group A streptococcus (GAS) causes a wide array of human diseases. Epidemiological picture of streptococcal infection in India is not complete. Hence, disease burden due to GAS in 5-15 yr old school children in northern India was studied and emm typing of GAS isolates was carried out to help in designing prevention strategies. METHODS: A cross-sectional survey was carried out among 4249 school children (5-15 yr) from Raipur Rani Block of Panchkula district in Haryana during 2000-2002; 334 children were followed up fortnightly for one year. Standard clinical and microbiological procedures were used for collection of swabs from throat and skin and confirmation of GAS and its emm types. RESULTS: Of the 4249 children studied, 658 (15.5%) had pharyngitis; 579 of them could be swabbed, of which 2.8 per cent had GAS. From 3591 children without pharyngitis, 3385 who could be swabbed, GAS was found in 1.3 per cent of them. Impetigo was rare (0.7%), but 7.1 per cent (2/28) children had GAS. In the followup study, 17.4 per cent (776/4447 child-contacts) had pharyngitis, 761 could be swabbed and 2.4 per cent had GAS; among those without pharyngitis, 2016 swabs could be taken and GAS was found in 1.3 per cent; whereas only 2.6 per cent (2/75) of skin sores had GAS. Three children had GAS pharyngitis twice during follow up. Fourteen different GAS emm types were found. emm 71, 77 and 81 constituted 69 per cent of the pharyngeal isolates. GAS pharyngitis and impetigo were more common in winters and summers respectively. INTERPRETATION & CONCLUSION: In north India, pharyngitis was more common than impetigo. Most prevalent emm types of GAS in this region differ from those included in M protein-based vaccines.

Virus isolation from wild-caught mosquitoes during a Japanese encephalitis outbreak in Kerala in 1996.

Out of 5357 wild-caught mosquitoes in 163 pools tested for virus using antigen capture ELISA and an insect-bioassay (inoculation into Toxorhynchites splendens larvae and identification by IFA using JE virus-specific monoclonal antibody), 16 flavivirus isolations were made of which 12 (75%) were identified as JE virus. Of the 12 JE virus isolations, 7 were from Culex tritaeniorhynchus, 3 from Mansonia uniformis and 1 each from Ma. indiana and Anopheles subpictus. Four isolations from Mansonia species for the first time reported here are noteworthy.

Rapid assessment procedures (RAP) for lymphatic filariasis.

BACKGROUND: New strategies are emerging for control of filariasis in terms of chemotherapy and vector control. Field application of these tools requires mapping and prioritization of filaria-endemic areas and quantification of the infection/ disease burden. Available procedures are time-consuming, costly and have poor sensitivity. Therefore, rapid assessment procedures need to be developed to assess the disease burden as well as monitor and evaluate control programmes. METHODS: Data collected on different variables from 25 areas in India and elsewhere were used. The relationship between prevalence of human infection and vector infection rate was analysed quantitatively. Due to lack of independent samples, only qualitative analysis was carried out between other epidemiological variables such as infection and disease prevalence in relation to age, gender and manifestation. RESULTS: There was a significant positive correlation between vector infection rate and infection prevalence in humans, suggesting that vector infection can be used as an indicator in the rapid assessment of infection prevalence. Scatter plots showed that community screening may be limited to the age group of 11-30 years for infection prevalence and 20-50 years for disease prevalence. Further, clinical surveys may be limited to only hydrocele prevalence which may be sufficient to predict the total disease. This can also be used as an alternative method by the community itself for delimiting endemic areas. CONCLUSIONS: Vector infection rate may be used as an indicator for rapid assessment of human infection. Alternatively, blood smear examination could be limited to the age group of 11-30 years. For a rapid survey of the diseases, males in the age group of 20-50 years could be examined only for hydrocele.

A novel 70-kDa Triton X-114-soluble antigen of Plasmodium falciparum that contains interspecies-conserved epitopes.

In order to identify novel conserved integral membrane and other membrane-associated proteins of Plasmodium falciparum, lambda gt11-P. falciparum DNA library phages were immunoscreened with convalescent-phase mouse sera and rabbit antiserum against Triton X-114-soluble proteins of P. falciparum. One recombinant phage clone, L857, reacted with both of the antibody probes. Insert DNA (857 bp long) in L857 was 69% dA+dT rich and hybridized to a fragment of 1800 bp from mung bean nuclease-digested P. falciparum genomic DNA. The cloned parasite DNA did not show notable sequence homology with any known protein gene. The L857-encoded polypeptide, p34 (M(r) 34 kDa) was expressed in bacteria, fused to glutathione S-transferase (GST). The fusion peptide, GST-p34 (M(r) 62 kDa), was recognized by immune serum against Triton X-114-soluble antigens of P. falciparum and was reactive with anti-P. falciparum, anti-Plasmodium yoelii, and anti-GST sera. Rabbit antiserum raised against the fusion peptide recognized a 70-kDa protein from lysates of P. falciparum cells and a putative homologous 100-kDa protein from lysates of P. yoelii. The rabbit serum anti-fusion peptide antibodies bound to acetone-fixed P. falciparum-infected erythrocytes and, in immunofluorescent antibody tests, produced a punctate pattern of fluorescence suggesting that the 70-kDa native protein is associated with an apical organelle of the parasite.

Dengue haemorrhagic fever and the dengue shock syndrome in India.

The clinical spectrum of dengue fever ranges from asymptomatic infection through severe haemorrhage and sudden fatal shock. Increased capillary permeability is the diagnostic feature of dengue haemorrhagic fever (DHF). The pathophysiology of DHF/dengue shock syndrome (DSS) is related to sequential infection with different serotypes of the virus, variations in virus virulence, interaction of the virus with environmental or host factors and a combination of various risk factors. Infection due to low virulence strains is assumed to be the reason for the infrequent incidence of serious dengue disease in India. Since all four serotypes of the dengue virus have been implicated in various outbreaks in this country and several outbreaks of DHF/DSS have been recorded since the first report in 1963, further epidemics of the disease are likely. The situation is aggravated by the recent emergence of DHF/DSS in Sri Lanka. In view of the potential of this disease to spread, effective preventive and control measures should be a priority.

Spread of lymphatic filariasis, re-emergence of leishmaniasis & threat of babesiosis in India.

The impact of unabated population growth and consequent ecological changes is felt in the spread of vector-borne diseases also. Filariasis, once considered to be more associated with urban areas and urbanization, is rapidly emerging as a major problem in rural areas. The need to delimit the endemic areas through a new rapid assessment procedure and the possible control methods of rural filariasis are discussed. Leishmaniasis has re-emerged with a vengeance in some parts of the country and the reasons for this are analyzed. Babesiosis, though zoonotic, has the potential to invade the domain of man in India.

Response of gravid Phlebotmus papatasi females to an oviposition attractant/stimulant associated with conspecific eggs.

Oviposition response of gravid P. papatasi females to conspecific eggs was studied in laboratory colonized sandflies. It was observed that significantly higher number of eggs were laid in the vicinity of conspecific eggs. However, a certain minimum number of eggs were required to be placed on the substratum to influence the rate of oviposition. The fecundity of females (mean = 56.5 +/- 4.9 eggs) exposed to conspecific eggs was significantly higher (P < 0.05) than that of blank control group. Perhaps, the chemical substances/pheromone of egg origin stimulated the oviposition rate. In an attempt to isolate the active ingredient, solvent washing of the conspecific eggs were tested. No increase in the rate of oviposition was noticed when the test site was treated with distilled water extract, whereas, significantly larger number of eggs were laid at the site treated with di-ethyl ether extract. This indicated that the oviposition attractant associated with the eggs dissolved in the organic solvent, but not in water. The possible application of this attractant for wild sandflies in nature and for regulating the site of oviposition on the substratum in the laboratory colonized sandflies needs to be explored.

A report on the demonstration of microfilariae of Brugia malayi in the brain of an experimental animal host, Mastomys natalensis.

The brain tissues of microfilaraemic animals, Mastomys natalensis, which were earlier inoculated (s.c.) with Brugia malayi infective larvae (100 each) were examined for the occurrence of Mf. This was done by staining squash preparations of the brain tissues which were cleared off from the vascular piamater. Animals with blood Mf count of 50 >/per 20 cu. mm were found to harbour Mf in the brain tissues. The Mf count in the brain varied from 5-86/81 cu. mm (sum of Mf detected in 3 tissue pieces, each of 27 cu. mm collected from 3 parts of the brain, viz., the cerebral hemispheres, cerebellum and medulla oblongata). Teh presence of Mf in the brain was confirmed by its detection in 20-micrometers-thick cryosections of the tissue. Also, fine needle aspirates of cerebral hemispheres of an animal showed live Mf.

Episodic adenolymphangitis and lymphoedema in patients with bancroftian filariasis.

In order to explore the relationship between acute and chronic disease, age-specific data on the frequency and duration of episodic adenolymphangitis (ADL) in patients with 3 defined grades of lymphoedema in bancroftian filariasis were examined. The age distribution of grades I and II exhibited a convex age profile, but that of grade III showed a monotonic increase. The mean duration of oedema increased with its grade (grade I, 0.3 years; grade III, 9.9 years). The mean number of ADL episodes in the previous year for all cases was 4.2 and it increased with grade (grade I, 2.4 and grade III, 6.2). The mean duration of each ADL episode for all cases was 4.1 d and it was independent of grade and age. The mean period lost to ADL episodes in the previous year was 17.5 d; it increased from 9.4 d with grade I to 28.5 d with grade III. The results imply that there is a dynamic progression through the grades of lymphoedema and that the frequency of ADL episodes is positively associated with this progression. However, the study design could not separate cause from effect.

Micro-spatial variation in filarial disease and risk of developing disease associated with microfilaremia in urban situation.

Clinical and parasitological surveys were carried out concurrently during 1986 in Pondicherry. The analyses showed that there was no significant micro-spatial variation in prevalence of total diseases (acute and chronic) and the manifestations such as hydrocele and lymphedema in the different zones and stations of Pondicherry urban area, a stable endemic area. Analyses on different filariometric indices in different stations showed a significant correlation between disease and mf prevalence (r = 0.4106; p = 0.037). The prevalence of disease and hydrocele in microfilaremic individuals (9.4% and 20.0% respectively) was higher compared to that observed in amicrofilaremic persons (6.4% and 11.2% respectively). The relative risk (RR) of parasite carriers developing disease (any manifestations) was marginally higher compared to amicrofilaremic persons (1.18). However, the RR of developing hydrocele manifestation due to microfilaremia was much greater (1.5) compared to amicrofilaremic persons. The attributable risk (AR) due to microfilaremia for developing hydrocele was 0.05. This suggests that although the risk is high in mf carriers, there might be alternate ways of developing disease without the infected person becoming microfilaremic. The limitations of point prevalence data on understanding complex dynamics of infection and disease are discussed.

Cumulative exposure and its relationship with chronic filarial disease in bancroftian filariasis.

Several hypotheses have been put forth about the factors influencing the dynamics of infection and disease in lymphatic filariasis. However, appropriate validation of these hypotheses by real situation analyses of epidemiological data is lacking. The present analyses examine the relationship between cumulative exposure to infection and prevalence of disease by utilizing the existing entomological and clinical data collected between 1981 and 1986 in Pondicherry, South India, endemic for bancroftian filariasis. While there was a significant negative association when the cumulative exposure was correlated with total prevalence of disease (r = 0.70, p = 0.024) as well as hydrocele alone (r = 0.74, p = 0.014), a significant positive association was found with prevalence of lymphedema (r = 0.72, p = 0.018). These results suggest that hydrocele development follows early after exposure, but prolonged exposure could result either in development of lymphedema or immune tolerance resulting in microfilaremia. These could also suggest that the pathomechanisms in development of hydrocele and lymphedema could follow different pathways. Implications of the present findings are discussed in light of the various hypotheses put forward by earlier studies.

Phlebotomine sandflies (Diptera: Psychodidae) of Tamil Nadu and Pondicherry, southern India, in relation to visceral leishmaniasis.

The Phlebotomine sandfly fauna of Tamil Nadu and Pondicherry in South India is described. Two morphologically different forms of Phlebotomus argentipes, the vector of Indian visceral leishmaniasis, are reported from Madras, a known focus of visceral leishmaniasis. A new species, Sergentomyia shettyi sp. nov., is described and Sergentomyia babu and Sergentomyia insularis stat. nov. are considered distinct species on the basis of their parapatric distribution. Phlebotomus papatasi and P. argentipes were the most abundant species around houses (37% and 29% of the catch, respectively), based on 1 year's sampling in villages near Pondicherry. The incidence of visceral leishmaniasis in Tamil Nadu since 1903 is summarized.

Estimation of permissible levels of transmission of bancroftian filariasis based on some entomological and parasitological results of a 5-year vector control programme.

The impact of a 5-year vector control programme on transmission of bancroftian filariasis and microfilaraemia levels in the human population was examined in Pondicherry, South India. The programme reduced and maintained the transmission of Wuchereria bancrofti at appreciably low levels in many localities of Pondicherry town, but its impact on overall microfilaraemia prevalence was only limited. However, prevalence of infection was reduced drastically in children born after establishment of the programme and only 18% of the number expected without vector control were found infected. No new infections were found in localities with less transmission intensity. It is proposed that a Transmission Intensity Index of 0.50 and Annual Transmission Potential of less than the 96-105 range are permissible levels of transmission, and below these levels no new infections may occur. The required duration of vector control to eliminate Culex-borne filariasis foci is estimated to be 11-12 years, slightly longer than that required to eradicate anopheline-borne filariasis.

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex.

Anopheles subpictus Grassi is shown to comprise four reproductively distinct species, designated A, B, C and D, occurring sympatrically in villages of Pondicherry, southeast India. Adult females were reared individually from wild larvae and examined for their morphological and chromosomal characters. Paracentric fixed inversions on the X-chromosome serve to distinguish the species cytogenetically, with no inversion heterozygotes (i.e. no interspecific hybrids) among totals of 717 species A (X+a, +b), 1863 species B (Xa, b), 869 species C (Xa, +b) and 1365 species D (X+a, b) identified. Morphologically, diagnostic characters for each of the four species are seen in the egg float ridge number, larval mesothoracic seta 4, pupal seta 7-I and the palpi of female adults. Species A, C and D immatures inhabit freshwater, whereas the malaria vector species B breeds in saltwater and was found only in coastal villages.