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An outbreak of cutaneous leishmaniasis in an Afghan refugee

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An outbreak of cutaneous leishmaniasis in an  Afghan refugee Powered By Docstoc
					TRANSACTIONS     OF THE ROYAL    SOCIETY    OF TROPICAL   MEDICINE     AND HYGIENE    (1999) 93,133-136


An outbreak of cutaneous                   leishmaniasis       in an Afghan          refugee settlement             in notth-
west Pakistan

Mark Rowland”*,      Arif Muniti,  Naeem Durrani’ , Harry Noyes and Hugh Reyburn’                ’ HealthNet International,
University Town, I? 0. Box 889, Peshawar, Pakistan; 2London School ofHygie ne and TropicalMedicine, London WClE 7HT,
UK; 3Natimal Institute of Health, Islamabad, Pakistan; 4Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK

                                                           Abstract
      Cutaneous leishmaniasis (CL) due to Leishmania tropica appears to be an emerging disease in parts of north-
      east Afghanistan and north-west Pakistan. Timargara, an Afghan refugee camp of 17 years’ standing, in the
      district of Dir, North West Frontier Province of Pakistan, experienced a major outbreak of CL in 1997 for
      the first time. As part of the investigation, each section of the camp was surveyed for CL. Around 38% of the
      9200 inhabitants bore active lesions and a further 13% had scars from earlier attacks. According to interview
      statements, 99% of earlier infections had healed within the previous 2 years. To confirm the diagnosis, a
      sample of current CL lesions was examined parasitologically. Amastigotes were detectable by microscopy in
      only 36% of lesions. However, 48% of slide-negative cases produced positive cultures and some cases
      negative to both microscopy and culture were positive by PCR. Overall detection rate was about 80%. The
      sandtly Phlebotomus ser.qenti, a known vector of L. tropica, was captured within the camn. indicating local
      transmission. CL has not been reported from this area-of Pakistan before. Although the iijority     of rekgees
      left Afghanistan 2 decades ago, cross-border movement ofmen is common. The Afhanistan caoital.-I Kabul.->
                                                                                                        I
      is cur&ntly experiencing a major epidemic of CL; infected migrant carriers fi-okKabul         are probably the
      source of the outbreak in Timargara.
      Keywords: cutaneous leishmaniasis, Leishmaniu rropica, epidemiology, refugees, Afghanistan, Pakistan, Phlebotomus
      sergenti


Introduction                                                         position of lesions, and any history of past CL were
   Cutaneous leishmaniasis (CL) due to Letihmania                    collected. The appearance of lesions was examined
(Leishmania) tropica appears to be an emerging disease               wherever possible.
in parts of north-east Afghanistan and north-west Paki-                  Second. a diaenostic confirmation of clinical CL was
stan. Recent epidemiological        data indicate that the           undertakin    using 3 techniques: microscopy, parasite
Afghanistan capital, Kabul, is experiencing a large in-              culture and polymerase chain reaction (PCR). Families
crease in CL (A. Brvceson, WHO/I-IN1            unpublished          of the original survey were selected using a random
assignment rep&t: Trkatmen; of cutaneous leishmaniasis in            number generator and the first 100 current cases of CL
Kabul, 4-15 December 1998; HEWITT et al., 1998).                     were invited to attend the camp’s health centre for
Refugee camps in north-west Pakistan are reporting                   diagnostic validation.
cases of CL for the first time (United Nations Hi&                      At the time of the diagnostic study, a number of light-
Commissioner      for Refugees, *HCR,           unpublished          traps were deployed in houses in the camp to identify
report, 1998).                                                       likely vectors of CL.
   The Timargara refugee camp in the district of Dir,                    Owing to logistical problems, there was a 7-week gap
north-west Pakistan, is a settled camp of 17 years’                  between the initial survey and the diagnostic study. None
standing. There are over 9000 residents, mostly from                 of the cases had received treatment as no treatment was
eastern, central, and northern Afghanistan. A few cases              available at the time.
of CL have been noticed over the past few years. How-                   Twenty cases were absent when called upon and 5
ever, in 1997-98 there was a large outbreak, causing                 refused to attend. These were replaced by the next 25
alarm within the refugee community and among the                     cases among the randomly selected families.
health authorities. CL also now occurs in neighbouring                   Cases were seen, their lesions counted and measured,
Pakistani villages.                                                  and the duration of disease was noted. The nodular
   As part of the investigation of the outbreak, the camp            margin of the most active-looking lesion was sliced open
was surveyed for CL and a sample of lesions was                      and a smear taken using a scalpel blade. Smears were
examined parasitologically       to confirm the diagnosis.           fixed in methanol, stained with Giemsa, and examined
Confirmation of this outbreak is important in the plan-              during the evening. Those with negative slides were
ning of public health strategies both in the camp and in             invited to re-attend the following day for a repeat smear
the local area. It also indicates the need for vigilance for         and culture sample, taken from another slit-skin pre-
emergent diseases associated with population move-                   paration. Twenty samples were also taken sequentially
ment.                                                                for PCR from the slide-negative cases (6 of which were
                                                                     found to be slide-positive on the second examination).
Methods                                                              All slides were examined by 2 experienced microscopists.
   There were 2 main comuonents to this studv.                       The microscopists each checked the findings of the other,
   First, the camp was surveyed in May 1997 bia team of              unaware of the first result.
resident communitv health workers (CHWsj trained in                     The samples for culture were inoculated into biphasic
the use of structures questionnaires ahd ide&ication      of         NNN media, transported on ice to the National Institute
CL lesions. Pairs of CHWs started in different sections of           of Health in Islamabad, and incubated according to the
the camp and selected households at random, using a                  method of EVANS et al. (1989). The solid phase of the
spinning bottle as a direction pointer. Sixty households             NNN media comnrised fresh. aseoticallv collected.
were visited and adults interviewed. Data on all residents,          defibrinated rabbit blood, mixeh wi& agar; NaCl, glul
the number of current cases of CL, the number and                    case, and gentamicin. The liquid phase comprised RPM1
                                                                     1640 medium with glutamine but without sodium
                                                                     bicarbonate, filtered through 0.22-p      disposable filters
Address for correspondence: Mark Rowland, Disease Control            to avoid bacterial contamination.
and Vector Biology Unit, London School of Hygiene and                   For PCR, samples were placed in 500 ~.LLof 4-M
Tropical Medicine, Keppel Street, London WClE 7HT, UK;               guanidine thiocyanate (GuSCN) and 0.25-M EDTA,
fax +44 (0) 171 580 9075, e-mail m.rowland@lshtm.ac.uk               and stored at 4°C until shipped by air to the Liverpool
134                                                                                                                    MARK   ROWLAND ETAL.


School of Tropical Medicine, UK. PCR was carried out                            was similar between boys (42%) and girls (43%) aged
as previously described by NOYES et al. (1998).                                 < 15 years, but was significantly lower among adult males
                                                                                (17%, n = 114) than among adult females (49%,
Results                                                                         n = 140). Adult males seem to be less at risk (relative
Surveys                                                                         risk: 0.35, x2 = 28, d.f. = 1, P <O.OOOl).
   According to interviews, most families surveyed (57%)                           The prevalence of active lesions increased with age up
originated from the Afghan eastern province of Kunar,                           to 15 years (Figure); the correlation was significant
 16% hailed from the northern provinces of Takhar and                           (r = O-77, P = 0.01). Older children seem to be more
Kundus, and others had come from other refugee camps                            at risk than any other age-group.
in the vicinity of Timargara.                                                      Sandfly light-traps were positioned indoors and out, in
   Over one-third, 304 (38%), of the 799 refugees                               the upper, middle, and lower parts of the camp. Phlebo-
surveyed had active lesions, and 106 (17%) reported                             tomus (Paraphlebotomus)     sergentiwere caught indoors and
scars and history characteristic of previous CL. All cases                      outdoors in each of the 6 traps (mean = 10 specimens
were recent: 87% of scars had healed within the past year                       per trap). No other species of phlebotomine was caught.
and 11% in the year before that. The mean number of
lesions per case was 2.2 (median 2, maximum 13). The                            Diagnostic     study
mean duration of active lesions was 5.3 months, and no                             Microscopy showed that only 36 of the 100 cases
lesion was reported to be older than 12 months.                                 sampled were positive for Leishmaniu amastigotes. Many
   The population of Timargara was very stable: 82% of                          of the lesions appeared to be healing. However, of the 50
families had lived there for 3 15 years, and only 2% had                        culture specimens taken from the 64 slide-negative cases,
moved in during the past 6 years. The average age was 17                        20 (48%) were positive for Leishmaniu and 8 were
years (median 10). Some two-thirds (61%) of the                                 discarded owing to bacterial or fungal contamination.
population had lived their entire lives in Pakistan.                            Fourteen slide-negative patients refused to re-attend for
   The Figure shows the prevalence of lesions and scars                         the second smear and culture (Table 1).
by age-group. Since an attack of CL gives life-long                                Nine (64%) of the 14 PCRs taken from slide-negative
immunity, this shows in effect the cumulative prevalence                        cases generated a PCR product of the same size as an L.
of CL with age. In endemic conditions cumulative                                tropica reference strain and distinct from L. infuntum and
prevalence tends to increase with age; in epidemic                              L. major strains. Restriction digests of the PCR product
conditions it is more constant across age-groups, as                            with Hue111 and HpaII produced fingerprint patterns
indicated here. Also shown in the Figure is the prevalence                      similar to L. tropica strains from Iran and central Pakistan
of active CL among the unscarred. Since a lesion lasts < 1                      (Balochistan province). All 6 of the slides that were
year this approximates to the current annual incidence                          parasite-positive on the second examination were also
risk. In fact, the prevalence of active lesions in the                          positive to PCR. Five samples were negative by micro-
unscarred was significantly greater in the age-poups                            scopy, PCR and culture (Table 2).
younger than 15 years (relative risk: 1.4, x = 15,                                 Ten specimens were sent for both PCR and culture; 3
d.f. = 1, P = O.OOOl), aswasthecumulativeprevalence.                            of the 5 culture-negative samples were positive to PCR
Further analysis reveals that prevalence of active lesions                      (Table 3).




                          0         10       20         30        40            60       60         70    so     90

                                                                         Age
                                         +cumu!ative prevalence Oactii     pfevalenw in unscarred

                              Figure. Prevalence     of cutaneous leishmaniasis at different ages (years).


                 Table 1. Comparison            of L. tropica      cases identified            by microscopy   and by cell
                 culture
                                                                                     Culture

                                                   Positive              Negative              Not done        Total
                 Microscopy
                   Positive                            3                        2                   31            36
                   Negative                           20                       22                   22            64
                   Total                              23                       24                   53           100
                                                                                                                                         135


                  Table 2. Comparison    of L. tropica          cases identified          by microscopy          and
                  polymerase chain reaction (PCR)
                                                                            PCR

                                               Positive        Negative           Not done              Total

                  ti!z;,“’                                         0
                    Negative                       8               z                 z:                     2
                    Total                         15                                 80                    100


Table 3. Comparison  of L. tro@ica         cases identified        tion (A. Bryceson, unpublished report, 1998; H. Rey-
by cell culture  and polymerase            chain reaction          bum, 1998, unpublished          surveys). As a result of
@‘CR)                                                              instability and civil war there is a regular flow of people,
                                                                   some of them infected, between Kabul and refugee
                                     PCR                           camps of Pakistan (I’. Marsden, unpublished report of
                                                                   the European Commission: Living in exile: coping strute-
                      Positive     Negative        Total           gihs of Afghan refugees, 1997). Adult male refugees are
                                                                   mobile in the search for work (I’. Marsden, unpublished
Culture                                                            report, 1997), and those from Timargara would visit
  Positive                             3                           Kabul or Parwan when travelling to the central or
  Negative               t             2                  z        northern Afghan provinces. Their frequent absence from
  Total                  5             5               10          the camp may explain why the prevalence of CL was
                                                                   lower in this group and why they appear less at risk.
                                                                      The diagnostic exercise described here would seem to
   The mean duration of disease was significantly longer           validate the findings of the initial survey. Although the
among cases negative to any diagnostic test (8 months,             proportion of slide-positive cases was very low (36%), 20
SD = 2.9) than for cases positive to any test (6.5 months,         of the 42 slide-negative cases sent for culture were
SD = 3.0) (Mann-Whitney          U-test, .a = -2.5, P =            positive (48%). By extrapolation, it would be expected
0.014). Similarly, the diameter of the largest lesion was          that 48% of the 64 slide-negative cases would have been
greater for cases negative to any test (mean 3.1 cm,               positive to culture, raising the rate ofpositive diagnosis to
SD = 2.3) than for cases positive to any test (2.4 cm,             76%. This rate might have been higher had there not
SD = 1.5) (a = 2.06, I’ = 0.039).                                  been a 7-week delay between the initial survey and the
                                                                   diagnostic study, during which perhaps 15% of cases
Discussion                                                         may have become negative to parasitological diagnosis.
   The survey of Timargara camp indicates a large out-             In addition 3 of the PCRs taken on 5 culture-negative
break of CL, with 38% of the population affected in 1997           cases were positive.       If this is similarly     extrapolated,     the
(and 13% in the year or so before that). The recent case           rate of positive diagnosis would have exceeded 80%.
histories, the population stability of the camp, and the           Cases negative to any of the parasitological tests were
presence of the vector sandfly I? sergenri (KILLICK-               likely   to be of longer    duration,     as successful     parasitolo-
KFNDRICK et al., 1994,1995) indicate that the outbreak             gical diagnosis of CL is known to be inversely related to
emanated from within the camp. The clinical appearance             the duration of the disease (RIPLEY & RIDLEY, 1983;
of the lesions was consistent with L. tropica infection, and       AZADEH et al., 1985).
L. tropica kDNA was detected in the lesions by PCR                     The occurrence of this large outbreak of CL in the
(NOYES et al., 1998) indicating that the CL is due to L.           Timargara refugee camp has important public health
tropica which is usually anthroponotic and urban.                  implications. CL may be carried by refugees into areas
   Older children seem to be more at risk than any other           previously unaffected by the disease (RAB et al., 1997).
age-group and men seem least at risk. Perhaps older                Epidemics may flare up with little warning if vector
children are less disciplined in covering up during the hot        sandflies are present, and l? sergenti, the vector of L.
summer nights. Women are required by their culture and             tropica, is widespread in Pakistan (NASIR, 1964; LEWIS,
religion to retain their modesty at all times, and use of           1967). Cases due to L. tropica may be persistent and
chadors (the clothing for covering the head) is common             disfiguring, and difficult and expensive to treat (A.
even at night.                                                     Bryceson, unpublished report, 1998). There is now a
    CL has not been reported from this part of Pakistan            trickle of cases reported from the local Pakistani popula-
before (RAB et al.. 1997). The outbreak mav have                   tion (UNHCR, unpublished report, 1998). Prompt and
resulted’from a single introduction, as the PCR evidence           effective measures will be required to prevent CL from
is of a single, homogeneous schizodeme clone in the 15             becoming endemic in the area.
cases examined (NOYES et al., 1998). CL in Pakistan due
to L. tropica commonly occurs in Multan in the southern            Acknowledgements
Punjab (BURNEY & LARI, 1986), in Balochistan province                  We are grateful to Musafa Khan and Fazle Rahim for the
where there was a major epidemic in the capital Quetta             microscopy, Hameed ur Rahman for the sandfly detection, and
after the earthquake of 1935 (MASSOOM & MARRI,                     Dr Fayez of the Timargara basic health unit for alerting us to the
 1993), and most recently in Azad Kashmir (RAB et al.,             outbreak. Rita Reybum assisted with the diagnostic survey. Dr
1997). Anthroponotic CL is more common in Afghani-                 Clive Davies and Professor Anthony Bryceson kindly comrnen-
stan; it is endemic to the southern city of Kandahar, the          ted on the manuscript. HealthNet International’s Malaria and
                                                                   Leishmaniasis Control Programme is supported by the Eur-
western city of Herat, and the central provinces of Kabul          opean Commission (DGl and ECHO), the United Nations
and Parwan (OMR et al., ~~~~;NADIM & ROSTAMI,                      High Commissioner        for Refugees, and Norwegian       Church Aid.
 1974; NADIM et al., 1979), but has not been reported
from the northern and eastern Afghan provinces from
which the majority of Timargara families originate. The            References
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