The spectrum of primary immunodeficiencies at a tertiary care hospital in Pakistan

Background Primary Immunodeficiency Disorders (PIDs) are well-known disorders in the West. but the recognition and diagnosis of these disorders is challenging in developing countries. We present the spectrum of PIDs seen at a tertiary care center in Pakistan, identified using clinical case definitions and molecular methods. Methods A retrospective chart review of children suspected to have PID was conducted at the Aga Khan University Hospital (AKUH) Karachi, Pakistan from 2010 to 2016. Data on demographics, clinical features, family history of consanguinity, sibling death, details of laboratory workup done for PID and molecular tests targeted panel next generation sequencing (NGS) or whole exome sequencing (WES) performed at the Geha laboratory at Boston Children’s Hospital, USA was collected. The study was exempted from the Ethical Review Committee of AKUH. Results A total of 43 children visited the hospital with suspected PID during the study period. Genetic testing was performed in 31/43 (72.1%) children. A confirmed diagnosis of PID was established in 20/43 (46.5%) children. A pathogenic gene variant was identified in 17(85%) of the 20 confirmed cases (Table 1). Twelve (60%) of the confirmed cases of PID were male. The most common presenting symptom was recurrent diarrhea 11/20 (55%). The mean (±S.D) age of the cases at the time of diagnosis was 4.2 (±4.1) years. Chronic granulomatous disease (CGD) was the most common 6/20 (30%) disorder, followed by severe combined immunodeficiency (SCID) 3/20 (15%), leukocyte adhesion deficiency (LAD) 3/20 (15%), agammaglobulinemia/hypogammaglobulinemia 3/20 (15%), and Hermansky-Pudlak Syndrome (HPS) 2/20 (10%). Wiskott-Aldrich Syndrome, Immunodeficiency Centromeric Instability and Facial Anomalies Syndrome (ICF 2), Trichohepatoenteric syndrome (TRES), and C3 deficiency were each diagnosed once {1/20 (4.3%) each} (Table 1). Of these 20 confirmed cases, almost all 19/20 (95%) had a family history of consanguinity. Sibling death was reported in 5/20 (25%) of these cases. Five out of the 20 (25%) children died over the 7-year period for various reasons. Conclusion PIDs are not uncommon in Pakistan; their diagnosis may be missed or delayed due to the overlapping of clinical features of PID with other diseases and a lack of diagnostic facilities. There is a need to build capacity for early recognition and diagnosis of PIDs to decrease morbidity and mortality.


INTRODUCTION
Primary immunodeficiency disorders (PIDs) are a heterogeneous group of genetic disorders characterized by an impaired ability of the immune system to produce a normal immune response. This is due to inherited defects in either cellular or humoral immunity, which results in a spectrum of issues such as recurrent infections, allergies, autoimmunity, and malignancies. 1,2 In neonates, PIDs often present with severe infections leading to death; whereas in adolescents these infections are less severe albeit recurrent. Diagnosing PIDs is challenging because of the variability in clinical presentation and limited availability of diagnostic tests, particularly in low middle-income countries (LMIC). When diagnostic tests are available, their cost often becomes a limiting factor. Advances in molecular diagnostic techniques and the identification of known gene defects have helped to facilitate the diagnosis of patients with PIDs. 3 The true global prevalence and distribution of PIDs remain unclear. The prevalence statistics available from nationwide registries are mostly derived from limited areas of the world. The data obtained from these registries often underestimate the true prevalence, because not all cases are reported to these registries, and due to ambiguity in what constitutes a PID case, some cases are missed. These issues are compounded in developing countries because of the lack of physician training in identification of these disorders and the limited access to diagnostics in these countries. Recent studies have shown that PIDs are more common than previously thought, and that around 1% of the population may have an underlying PID. 4 The burden of PID varies by region, being highest in the United States of America (USA), followed by Europe, Latin America, Middle East, Asia, and finally Africa. 4 This frequency may be biased by the availability of resources for diagnosis of these disorders.
Most PIDs are autosomal recessive, which makes it safe to assume that the incidence of PIDs is greater in regions having higher rates of consanguinity. However, limited studies have been carried out in such regions and hence the burden and type of PIDs in such areas is relatively unknown.
Consanguineous marriages are common in developing Asian countries ranging between 20 and 70% of all marriages, and Pakistan is no different, with about 70% of the marriages being consanguineous. [5][6][7] The data on PIDs in Pakistan is currently confined to a few case reports, case series, and editorials; there are no detailed reports on the spectrum of PIDs seen in the country. [8][9][10][11] Diagnosing and treating PIDs is a challenge in Pakistan. Patients often die before the disorder is recognized by a physician, leading to a delay in diagnosis or death of the child. Families are often referred to tertiary care centers following the death of a sibling or for admission for life-threatening infections. Even in large tertiary care centers with well-equipped laboratories, there are limited diagnostic facilities available for confirming PIDs. Even when these are available, the diagnostic tests are expensive and beyond the reach of most families as the health care system of Pakistan relies on out-of-pocket payments for health expenditures. Once the diagnosis of PID is confirmed, supportive therapies like antibiotic and antifungal prophylaxis, intravenous (I/V), or subcutaneous (S/ C) immunoglobulin therapy, depending on the underlying disorder, are offered. Intravenous immunoglobulin (IVIG) is the standard therapy for most humoral deficiencies, but it is expensive and beyond the reach of most patients in low middle income countries (LMICs). S/C immunoglobulins provide ease of administration, but their availability is limited in LMICs. Bone marrow transplant is available in certain settings but has its limitations in countries like Pakistan where no donor registries are available, and the cost of the transplant coupled with the risk of infections in the posttransplant period make this option challenging to pursue. Gene therapy is another promising albeit costly treatment option undergoing experimentation and is now considered an option for the treatment of multiple non-life-threatening disorders (ie, immunological disorders and systemic protein deficiency). This study aims to report the spectrum of PID cases observed at a tertiary care center in Pakistan, with a focus on the molecular diagnosis in this patient population over the last 7 years.

MATERIALS AND METHODS
The Aga Khan University Hospital (AKUH) is a 700 bed, not-for-profit tertiary care hospital in Karachi, Pakistan. The hospital receives referrals from all over the country. A retrospective chart review of children suspected to have PID was conducted at the AKUH, Pakistan from 2010 to 2016. Data on demographics, clinical features, age at onset of symptoms, age at presentation, history of recurrent infections, skin allergies, family history of consanguinity, sibling death, details related to laboratory workup done for PID (complete blood count, serum immunoglobulin levels, nitrotetrazolium blue test (NBT), dihydrorhodamine(DHR) and flow cytometric analysis) were collected. Molecular tests targeted panel next generation sequencing (NGS) or whole exome sequencing (WES) were performed at Geha laboratory at Boston Children's Hospital, USA. Targeted panel NGS was performed using the PID v2 panel and Ion TorrentÔ S5 sequencer (Ther-moFisher), with an average coverage of 328x. Variant annotation was performed with VarSeqÔ software (Golden Helix). Whole exome sequencing (WES) was performed using a previously described pipeline with an average on-target coverage of 80Â. 12 The cost of shipping and molecular testing was covered by a philanthropic grant from the Perkins Fund. Data were analyzed using IBM SPSS Statistics v. 20.0. Mean AE S.D is reported for quantitative variable like age at diagnosis, delay in diagnosis (days), etc. Frequency and percentages are reported for categorical variables such as gender, type of PID, clinical features, history of sibling death, consanguinity in family, mortality, etc.

DISCUSSION
In this case series, 85% of the children were able to get a molecular diagnosis through the support of Boston Children's Hospital. CGD was the commonest disorder observed. This is consistent with data reported from other Asian countries in which phagocytic defects were the most common type of PIDs ranging from 29% to 60%. [13][14][15] Almost all cases in this study had a family history of consanguinity. Death of siblings was seen in 25% of the cases. Out of the children who died during the study period, 80% of them were diagnosed after they had died.
Due to a lack of facilities, diagnosing PIDs continues to be a challenge in developing countries. Partnering with international organizations is Patients with PIDs are often diagnosed based on a clinical history of recurrent infections due to atypical or less virulent pathogens. 16 However, they can also present with non-infectious manifestations, such as autoimmune disease, or albinism. 17 Verma et al published a case series of 27 PID cases in whom recurrent pneumonia and recurrent diarrhea were the most common presentations. In our case series, recurrent diarrhea was the most common clinical presentation followed by recurrent pneumonia, oral thrush, skin rash, and abscesses. 18 Live vaccines (eg, BCG, OPV) should not be administered to children with PIDs due to the risk of developing mycobacterial disease with the bacillus CalmetteÀGuérin (BCG) vaccine or Vaccine Derived Poliovirus (VDP) with the oral polio virus vaccine (OPV). 19 Shahmohammadi S et al reviewed 17 cases of disseminated BCG infection in Iran from 2005 to 2010 and observed that 10 (59%) out of the 17 cases had impaired immunity due to an incompetent immune system. 20 In Pakistan, these vaccines (BCG and OPV) are a part of the national immunization programme; hence, they are administered routinely, much earlier before a diagnosis of immunodeficiency can be suspected or established in children. In our case series, almost all cases were vaccinated with the BCG and OPV at birth; so far, none of them have developed any sign of disease due to the vaccination (VDP or Tuberculosis). Although this report is based on a limited number of cases, it does raise the need for a newborn screening program in the long term, so that children suspected of having a PID do not receive a live vaccine at birth.
Early recognition and prompt diagnosis of PIDs help in preventing significant disease related morbidity and mortality. A clinical history and physical examination together with supportive laboratory investigations can provide clues about an underlying PID. Nevertheless, molecular testing plays an essential role in not only confirming the diagnosis but also in identifying the exact nature of the defect. It also assists in patient care as it allows for prognostic counseling. 21 Varying degree of support for diagnosing PIDs is available from different institutions and philanthropic foundations such as the (International Union of Immunological Sciences (IUIS), European Society for Immunodeficiencies (ESID), Immune Deficiency Foundation (IDF), and Jeffrey Modell Foundation JMF). In most LMICs, diagnostic and therapeutic services are sparse and/or inaccessible. Armed Forces Institute of Pathology (AFIP) in Rawalpindi, Pakistan is a center where limited immune diagnostics are available but molecular techniques are still lacking. Furthermore, this is the only center where some form of diagnostics is available for PIDs. For a country with a population of 200 million and a birth rate of 29.8 births per 1,000 people, a single facility is not enough to meet the demands.
To our knowledge, this is the first report of PID cases from Pakistan confirmed with molecular diagnosis. The cases reported here only reflect cases referred to a large tertiary care center and represent only the tip of the iceberg.
As a next step, we plan to set up a national registry to bring together individual efforts so that medical care for affected individuals can be improved. Having a national registry will facilitate exchange of experience in the diagnosis and management of PIDs with international registries and will help push policies for support of children with an identified PID. We plan to work towards achieving philanthropic support for IVIG/SCIG or bone marrow transplant (BMT) for the treatment of diagnosed patients. Hopefully, these collective efforts will help with early identification of PIDs, aid in tracking and establishing epidemiological data of PIDs in Pakistan, and help decrease the mortality of patients in developing countries around the world.

CONCLUSION
PIDs are not uncommon in Pakistan; their diagnosis may be missed or delayed due to the overlapping of clinical features of PIDs with other diseases and a lack of diagnostic facilities. There is a need to increase testing capacity for early recognition, diagnosis and management of PIDs to decrease morbidity and mortality. There is also a need to establish a national registry for estimating the true burden of PIDs in the country to aid in policy recommendations.