The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

BACKGROUND The risk for emotional and behavioral problems is known to be high among children of depressed mothers, but little is known about the impact of prenatal and postnatal depression on the physical health of infants. OBJECTIVE To determine whether maternal depression is a risk factor for malnutrition and illness in infants living in a low-income country. DESIGN Prospective cohort study. SETTING Rural community in Rawalpindi, Pakistan. PARTICIPANTS Six hundred thirty-two physically healthy women were assessed in their third trimester of pregnancy to obtain at birth a cohort of 160 infants of depressed mothers and 160 infants of psychologically well mothers. MAIN OUTCOME MEASURES All infants were weighed and measured at birth and at 2, 6, and 12 months of age, and they were monitored for episodes of diarrhea and acute respiratory infections. The mothers' mental states were reassessed at 2, 6, and 12 months. Data were collected on potential confounders of infant outcomes, such as birth weight and socioeconomic status. RESULTS Infants of prenatally depressed mothers showed significantly more growth retardation than controls at all time points. The relative risks for being underweight (weight-for-age z score of less than -2) were 4.0 (95% confidence interval [CI], 2.1 to 7.7) at 6 months of age and 2.6 (95% CI, 1.7 to 4.1) at 12 months of age, and the relative risks for stunting (length-for-age z score of less than -2) were 4.4 (95% CI, 1.7 to 11.4) at 6 months of age and 2.5 (95% CI, 1.6 to 4.0) at 12 months of age. The relative risk for 5 or more diarrheal episodes per year was 2.4 (95% CI, 1.7 to 3.3). Chronic depression carried a greater risk for poor outcome than episodic depression. The associations remained significant after adjustment for confounders by multivariate analyses. CONCLUSIONS Maternal depression in the prenatal and postnatal periods predicts poorer growth and higher risk of diarrhea in a community sample of infants. As depression can be identified relatively easily, it could be an important marker for a high-risk infant group. Early treatment of prenatal and postnatal depression could benefit not only the mother's mental health but also the infant's physical health and development.

Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.

HASH(0xd360bd0)

The Oriental white-backed vulture (OWBV; Gyps bengalensis) was once one of the most common raptors in the Indian subcontinent. A population decline of >95%, starting in the 1990s, was first noted at Keoladeo National Park, India. Since then, catastrophic declines, also involving Gyps indicus and Gyps tenuirostris, have continued to be reported across the subcontinent. Consequently these vultures are now listed as critically endangered by BirdLife International. In 2000, the Peregrine Fund initiated its Asian Vulture Crisis Project with the Ornithological Society of Pakistan, establishing study sites at 16 OWBV colonies in the Kasur, Khanewal and Muzaffargarh-Layyah Districts of Pakistan to measure mortality at over 2,400 active nest sites. Between 2000 and 2003, high annual adult and subadult mortality (5-86%) and resulting population declines (34-95%)(ref. 5 and M.G., manuscript in preparation) were associated with renal failure and visceral gout. Here, we provide results that directly correlate residues of the anti-inflammatory drug diclofenac with renal failure. Diclofenac residues and renal disease were reproduced experimentally in OWBVs by direct oral exposure and through feeding vultures diclofenac-treated livestock. We propose that residues of veterinary diclofenac are responsible for the OWBV decline.

To determine the prevalence and routes of transmission of hepatitis C virus (HCV) infection in Hafizabad, Pakistan, we collected sera in 1993 from a geographically based random sample of residents, and in 1994 identified 15 HCV-infected individuals (cases) and 67 age and sex matched uninfected individuals (controls). Initially we approached 504 households, and collected serum from a randomly selected household member in 309 (64%). Twenty persons (6.5%) had anti-HCV antibody; 31% percent had hepatitis B core antibodies, and 4.3% had hepatitis B surface antigen. In the case-control study, persons who received more therapeutic injections (categorized as averaging 1, 2-4, 5-9 or > 10 injections per year in the previous 10 years) were more likely to be infected with HCV (odds ratio 0, 1.5, 2.5 and 6.9 respectively, P = 0.008) compared to persons averaging 0 injections per year. Efforts to limit therapeutic injections to only those that are medically indicated and that use sterile equipment are essential in order to prevent transmission of HCV.

BACKGROUND There are approximately 4 million neonatal deaths and half a million maternal deaths worldwide each year. There is limited evidence from clinical trials to guide the development of effective maternity services in developing countries. METHODS We performed a cluster-randomized, controlled trial involving seven subdistricts (talukas) of a rural district in Pakistan. In three talukas randomly assigned to the intervention group, traditional birth attendants were trained and issued disposable delivery kits; Lady Health Workers linked traditional birth attendants with established services and documented processes and outcomes; and obstetrical teams provided outreach clinics for antenatal care. Women in the four control talukas received usual care. The primary outcome measures were perinatal and maternal mortality. RESULTS Of the estimated number of eligible women in the seven talukas, 10,114 (84.3 percent) were recruited in the three intervention talukas, and 9443 (78.7 percent) in the four control talukas. In the intervention group, 9184 women (90.8 percent) received antenatal care by trained traditional birth attendants, 1634 women (16.2 percent) were seen antenatally at least once by the obstetrical teams, and 8172 safe-delivery kits were used. As compared with the control talukas, the intervention talukas had a cluster-adjusted odds ratio for perinatal death of 0.70 (95 percent confidence interval, 0.59 to 0.82) and for maternal mortality of 0.74 (95 percent confidence interval, 0.45 to 1.23). CONCLUSIONS Training traditional birth attendants and integrating them into an improved health care system were achievable and effective in reducing perinatal mortality. This model could result in large improvements in perinatal and maternal health in developing countries.

Crimean-Congo Haemorrhagic Fever (CCHF) is an often-lethal haemorrhagic fever caused by a tick-borne virus. There are no published data on ribavirin treatment of CCHF-infected patients, despite established in-vitro and in-vivo sensitivity. We report three health workers--two surgeons and a hospital worker--infected with CCHF virus in Pakistan who were treated with oral ribavirin 4 g/day for four days, then 2.4 g/day for six days. Intravenous ribavirin was unavailable. All three patients were severely ill with low platelet and white-cell counts, raised aspartate transaminase and evidence of impaired haemostasis. Based on published reports, all had an estimated probability of death of 90% or more. The patients became afebrile, and their haematological and biochemical abnormalities returned to normal within 48 h of ribavirin treatment; all made a complete recovery, and developed IgG and IgM antibody to CCHF virus. Our experience with ribavirin treatment is encouraging, but does not constitute evidence of efficacy. Given the difficulties in gathering adequate treatment data, we propose a consensus protocol for both intravenous and oral treatment of CCHF. This protocol could be distributed to key medical personnel in areas endemic for CCHF and used to provide a firm basis for effective treatment recommendations.

BACKGROUND The high prevalence of depression in developing countries is not well understood. This study aimed to replicate the previous finding of a high prevalence of depression in Pakistan and assess in detail the associated social difficulties. METHOD A two-phase survey of a general population sample in a Pakistani village was performed. The first-phase screen used the Personal Health Questionnaire (PHQ) and the self-rating questionnaire (SRQ). A one in two sample of high scorers and a one in three sample of the low scorers were interviewed using the Psychiatric Assessment Schedule (PAS) and Life Events and Difficulties Schedule (LEDS). RESULTS A total of 259 people were screened (96% response rate). The second stage yielded 55 cases, of whom 54 had depressive disorder, and 48 non-cases. The adjusted prevalence of depressive disorders was 44-4%(95% CI 35.3 to 53.6): 25.5% in males and 57.5% in females. Nearly all cases had lasted longer than 1 year. Comparison of the cases and non-cases indicated that cases were less well educated, had more children and experienced more marked, independent chronic difficulties. Multivariate analysis indicated that severe financial and housing difficulties, large number of children and low educational level were particularly closely associated with depression. CONCLUSION This study confirms the high prevalence of depressive disorders in Pakistan and suggests that this may be higher than other developing countries because of the high proportion of the population who experience social adversity.

Following reports of frequent deaths associated with jaundice and chronic liver disease among adults in a periurban community of Karachi, Pakistan, an investigation was conducted to evaluate the relationship between injections and viral hepatitis infections, to identify the reasons why patients received frequent injections, and to observe the injection practices employed in clinics. Two hundred and three adult patients were interviewed as they left each of the 18 area clinics. Practitioners were interviewed and three consecutive injections were observed at each clinic. Eighty-one per cent of patients received an injection on the day of the interview. Of the 135 patients who provided a serum sample, 59 (44%) had antibodies against hepatitis C virus and 26 (19%) had antibodies against hepatitis B virus. Patients who received more injections were more likely to be infected with hepatitis C. If oral and injected medications were equally effective, 44% of patients preferred injected medication. None of the practitioners knew that hepatitis C could be transmitted by injections. Non-sterile syringes and needles that had been used earlier in the day on other patients were used for 94% of the observed injections. Interventions to limit injections to those which are safe and clinically indicated are needed to prevent injection-associated infections in Pakistan and other low-income countries.